1,3-propanediol and polymer derivatives from a fermentable carbon source

ABSTRACT

A new polypropylene terephthalate composition is provided. The polypropylene terephthalate is comprised of 1,3-propanediol and terephthalate. The 1,3-propanediol is produced by the bioconversion of a fermentatble carbon source, preferable glucose. The resulting polypropylene terephthalate is distinguished from petrochemically produced polymer on the basis of dual carbon-isotopic fingerprinting which indicates both the source and the age of the carbon.

FIELD OF INVENTION

[0001] The invention relates to a new 1,3-propanediol monomer andpolymers derived from these monomers. More specifically, polypropyleneterephthalate has been produced from a 1,3-propanediol monomer preparedby bioconverting a fermentable carbon source directly to 1,3-propanediolusing a single microorganism.

BACKGROUND

[0002] 1,3-Propanediol is a monomer useful in the production ofpolyester fibers and in the manufacture of polyurethanes.

[0003] It has been known for over a century that 1,3-propanediol can beproduced from the fermentation of glycerol. Bacterial strains able toproduce 1,3-propanediol have been found, for example, in the groupsCitrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella,Lactobacillus, and Pelobacter. In each case studied, glycerol isconverted to 1,3-propanediol in a two step, enzyme-catalyzed reactionsequence. In the first step, a dehydratase catalyzes the conversion ofglycerol to 3-hydroxypropionaldehyde (3-HP) and water, Equation 1. Inthe second step, 3-HP is reduced to 1,3-propanediol by a NAD⁺-linkedoxidoreductase, Equation 2. The 1,3-propanediol is not metabolizedfurther and, as a result,

Glycerol→3-HP+H₂O  (Equation 1)

3-HP+NADH+H⁺→1,3-Propanediol+NAD⁺  (Equation 2)

[0004] accumulates in high concentration in the media. The overallreaction consumes a reducing equivalent in the form of a cofactor,reduced β-nicotinamide adenine dinucleotide (NADH), which is oxidized tonicotinamide adenine dinucleotide (NAD⁺).

[0005] The production of 1,3-propanediol from glycerol is generallyperformed under anaerobic conditions using glycerol as the sole carbonsource and in the absence of other exogenous reducing equivalentacceptors. Under these conditions in e.g., strains of Citrobacter,Clostridium, and Klebsiella, a parallel pathway for glycerol operateswhich first involves oxidation of glycerol to dihydroxyacetone (DHA) bya NAD⁺-(or NADP⁺-) linked glycerol dehydrogenase, Equation 3. The DHA,following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHAkinase (Equation 4),

Glycerol+NAD⁺→DHA+NADH+H⁺  (Equation 3)

DHA+ATP→DHAP+ADP  (Equation 4)

[0006] becomes available for biosynthesis and for supporting ATPgeneration via e.g., glycolysis. In contrast to the 1,3-propanediolpathway, this pathway may provide carbon and energy to the cell andproduces rather than consumes NADH.

[0007] In Klebsiella pneumoniae and Citrobacter freundii, the genesencoding the functionally linked activities of glycerol dehydratase(dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase(dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dharegulon. The dha regulons from Citrobacter and Klebsiella have beenexpressed in Escherichia coli and have been shown to convert glycerol to1,3-propanediol.

[0008] Although biological methods of both glycerol and 1,3-propanediolproduction are known, it has never been demonstrated that the entireprocess can be accomplished by a single organism.

[0009] Neither the chemical nor biological methods described above forthe production of 1,3-propanediol is well suited for industrial scaleproduction. This is because the chemical processes are energy intensiveand the biological processes require glycerol, an expensive startingmaterial. A method requiring low energy input and an inexpensivestarting material is needed. A more desirable process would incorporatea microorganism that would have the ability to convert basic carbonsources, such as carbohydrates or sugars, to the desired 1,3-propanediolend-product.

[0010] There are several difficulties that are encountered whenattempting to biologically producte 1,3-propanediol by a single organismfrom an inexpensive carbon substrate such as glucose or other sugars.The biological production of 1,3-propanediol requires glycerol as asubstrate for a two-step sequential reaction in which a dehydrataseenzyme (typically a coenzyme B₁₂-dependent dehydratase) convertsglycerol to an intermediate, 3-hydroxypropionaldehyde, which is thenreduced to 1,3-propanediol by a NADH- (or NADPH) dependentoxidoreductase. The complexity of the cofactor requirements necessitatesthe use of a whole cell catalyst for an industrial process whichutilizes this reaction sequence for the production of 1,3-propanediol.Furthermore, in order to make the process economically viable, a lessexpensive feedstock than glycerol or dihydroxyacetone is needed. Glucoseand other carbohydrates are suitable substrates, but, as discussedabove, are known to interfere with 1,3-propanediol production.

SUMMARY OF THE INVENTION

[0011] The present invention provides a 1,3-propanediol composition ofmatter produced by the process comprising the bioconversion of a carbonsubstrate, other than glycerol or dehydroxy acetone dihydroxyacetone, to1,3-propanediol by a single microorganism having at least one gene thatexpresses a dehydratase enzyme by contacting said microorganism withsaid substrate.

[0012] The invention further provides a biosourced 1,3-propanediolcomposition of matter having δ¹³C of about −10.9 to about −15.4,preferably about −13.22 to about −14.54, and most preferably about−13.84 to about −13.92, and a f_(M) ¹⁴C of about 1.04 to about 1.18,preferably about 1.106 to about 1.129, and most preferably about 1.111to about 1.124.

[0013] Additionally the invention provides a polymer comprising at leasttwo repeating units of biosourced 1,3-propanediol, characterized by aδ¹³C of −10.74 to about −17.02, preferably about −13.22 to about −14.54,and most preferably about −13.84 to about −13.82 to about −13.94, and af_(M) ¹⁴C of about 1.003 to about 1.232, preferably about 1.106 to about1.129, and most preferably about 1.111 to about 1.124.

[0014] In another embodiment, the invention provides a polymercomprising at least two repeating units of biosourced polypropyleneterephthalate, characterized by a δ¹³C of about −24.74 to about −24.88,and a f_(M) ¹⁴C of about 0.299 to about 0.309 and a polymeric unitconsisting of polypropylene terephthalate having a δ¹³C of about −24.74to about −24.88, and a f_(M) ¹⁴C of about 0.299 to about 0.309.

[0015] In another embodiment the invention provides a method foridentifying the presence of a biosourced 1,3-propanediol in a sample,the method comprising (a) purifying the 1,3-propanediol from the sample;and (b) determining the δ¹³C and f_(M) ¹⁴C characterizing the sample ofstep (a), wherein a δ¹³C of about −10.9 to about −15.4 and a f_(M) ¹⁴Cof about 1.04 to about 1.18 indicates the presence of a biosourced1,3-propanediol. Additionally, the specific source of biosourced carbon(e.g. glucose or gycerol) can be ascertained by dual carbon-isotopicanalysis.

[0016] Finally, the invention provides an article of manufacturecomprising the described composition produced by the process and in aform selected from the group consisting of a film, a fiber, a particle,and a molded article.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 shows the dual isotope diagram of the data in Table 1. Theabscissa gives the values of δ¹³C (per mil) referenced to PDB. Theordinate gives ¹⁴C expressed as f_(M) (fraction of modern).

[0018]FIG. 2, an expanded view of FIG. 1, shows the individual valuesfor the two polypropylene terephthalate samples prepared with biosourced1,3-propanediol. Error bars reflect the standard uncertainties for eachisotope.

[0019]FIG. 3, an expanded view of FIG. 1, shows the individual valuesfor the two biosourced 1,3-propanediol samples. Error bars reflect thestandard uncertainties for each isotope.

[0020]FIG. 4, an expanded view of FIG. 1, shows the individual valuesfor the two glucose samples. Error bars reflect the standarduncertainties for each isotope.

[0021]FIG. 5 shows ¹⁴C precision data for selected samples.

[0022]FIG. 6 shows ¹³C precision data for selected samples.

BRIEF DESCRIPTION OF BIOLOGICAL DEPOSITS

[0023] The transformed E. coli DH5α containing cosmid pKP1 containing aportion of the Klebsiella genome encoding the glycerol dehydrataseenzyme was deposited on Apr. 18, 1995 with the ATCC under the terms ofthe Budapest Treaty and is identified by the ATCC number ATCC 69789. Thetransformed E. coli DH5α containing cosmid pKP4 containing a portion ofthe Klebsiella genome encoding a diol dehydratase enzyme was depositedon Apr. 18, 1995 with the ATCC under the terms of the Budapest Treatyand is identified by the ATCC number ATCC 69790. As used herein, “ATCC”refers to the American Type Culture Collection international depositorylocated at 10801 University Boulevard, Manassas, Va., 20110-2209, U.S.A.The “ATCC No.” is the accession number to cultures on deposit with theATCC.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention describes the bioconversion of afermentable carbon source for the production of 1,3-propanediol from asingle microorganism harboring a dehydratase enzyme. The methodincorporates a microorganism containing a dehydratase enzyme which iscontacted with a fermentable carbon substrate and 1,3-propanediol isisolated from the growth media. The single organism may be a wild-typeorganism or may be a genetically altered organism harboring a geneencoding a dehydratase enzyme. The invention further provides newmonomers and polymers derived from the biosourced 1,3-propanediol.

[0025] Applicants have solved the stated problem and the presentinvention provides for bioconverting a fermentable carbon sourcedirectly to 1,3-propanediol using a single organism. Glucose is used asa model substrate and the bioconversion is applicable to any existingmicroorganism. Microorganisms harboring the gene for a dehydratase areable to convert glucose and other sugars through the glyceroldegradation pathway to 1,3-propanediol with good yields andselectivities. Furthermore, the present invention may be generallyapplied to include any carbon substrate that is readily converted toglycerol, dihydroxyacetone, or C₃ compounds at the oxidation state ofglycerol (e.g., glycerol 3-phosphate) or dihydroxyacetone (e.g.,dihydroxyacetone phosphate or glyceraldehyde 3-phosphate).

[0026] Biologically produced 1,3-propanediol represents a new feedstockfor useful polymers, such as 1,3-propanediol polyol and polypropyleneterephthalate. Polypropylene terephthalate has not previously beenproduced from a biosourced monomer. As such, it is a new composition ofmatter, comprising terephthalate derived from petrochemical sources and1,3-propane-diol derived from biosourced carbon substrates other thanglycerol and dihydroxyacetone. This new polymer may be distinguishedfrom polymer derived from all petrochemical carbon on the basis of dualcarbon-isotopic fingerprinting. Additionally, the specific source ofbiosourced carbon (e.g. glucose vs. glycerol) can be determined by dualcarbon-isotopic fingerprinting.

[0027] This method usefully distinguishes chemically-identicalmaterials, and apportions carbon in the copolymer by source (andpossibly year) of growth of the biospheric (plant) component. Theisotopes, ¹⁴C and ¹³C, bring complementary information to this problem.The radiocarbon dating isotope (¹⁴C), with its nuclear half life of 5730years, clearly allows one to apportion specimen carbon between fossil(“dead”) and biospheric (“alive”) feedstocks [Currie, L. A. “SourceApportionment of Atmospheric Particles,” Characterization ofEnvironmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 ofVol. I of the IUPAC Environmental Analytical Chemistry Series (LewisPublishers, Inc) (1992) 3-74]. The basic assumption in radiocarbondating is that the constancy of ¹⁴C concentration in the atmosphereleads to the constancy of ¹⁴C in living organisms. When dealing with anisolated sample, the age of a sample can be deduced approximately by therelationship

t=(−5730/0.693)ln(A/A_(O))  (Equation 5)

[0028] where t=age, 5730 years is the half-life of radiocarbon, and Aand A_(O) are the specific ¹⁴C activity of the sample and of the modernstandard, respectively [Hsieh, Y., Soil Sci. Soc. Am J., 56, 460,(1992)]. However, because of atmospheric nuclear testing since 1950 andthe burning of fossil fuel since 1850, ¹⁴C has acquired a second,geochemical time characteristic. Its concentration in atmosphericCO₂—and hence in the living biosphere—approximately doubled at the peakof nuclear testing, in the mid-1960s. It has since been graduallyreturning to the steady-state cosmogenic (atmospheric) baseline isotoperate (¹⁴C/¹²C) of ca. 1.2×10⁻¹², with an approximate relaxation“half-life” of 7-10 years. (This latter half-life must not be takenliterally; rather, one must use the detailed atmospheric nuclearinput/decay function to trace the variation of atmospheric andbiospheric ¹⁴C since the onset of the nuclear age.) It is this latterbiospheric ¹⁴C time characteristic that holds out the promise of annualdating of recent biospheric carbon. ¹⁴C can be measured by acceleratormass spectrometry (AMS), with results given in units of “fraction ofmodern carbon” (f_(M)). f_(M) is defined by National Institute ofStandards and Technology (NIST) Standard Reference Materials (SRMs)4990B and 4990C, known as oxalic acids standards HOxI and HOxII,respectively. The fundamental definition relates to 0.95 times the¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughlyequivalent to decay-corrected pre-Industrial Revolution wood. For thecurrent living biosphere (plant material), f_(M)≈1.1.

[0029] The stable carbon isotope ratio (¹³C/¹²C) provides acomplementary route to source discrimination and apportionment. The¹³C/¹²C ratio in a given biosourced material is a consequence of the¹³C/¹²C ratio in atmospheric carbon dioxide at the time the carbondioxide is fixed and also reflects the precise metabolic pathway.Regional variations also occur. Petroleum, C₃ plants (the broadleaf), C₄plants (the grasses), and marine carbonates all show significantdifferences in ¹³C/¹²C and the corresponding δ¹³C values. Furthermore,lipid matter of C₃ and C₄ plants analyze differently than materialsderived from the carbohydrate components of the same plants as aconsequence of the metabolic pathway. Within the precision ofmeasurement, ¹³C shows large variations due to isotopic fractionationeffects, the most significant of which for the instant invention is thephotosynthetic mechanism. The major cause of differences in the carbonisotope ratio in plants is closely associated with differences in thepathway of photosynthetic carbon metabolism in the plants, particularlythe reaction occurring during the primary carboxylation, i.e., theinitial fixation of atmospheric CO₂. Two large classes of vegetation arethose that incorporate the “C₃” (or Calvin-Benson) photosynthetic cycleand those that incorporate the “C₄” (or Hatch-Slack) photosyntheticcycle. C₃ plants, such as hardwoods and conifers, are dominant in thetemperate climate zones. In C₃ plants, the primary CO₂ fixation orcarboxylation reaction involves the enzyme ribulose-1,5-diphosphatecarboxylase and the first stable product is a 3-carbon compound. C₄plants, on the other hand, include such plants as tropical grasses, cornand sugar cane. In C₄ plants, an additional carboxylation reactioninvolving another enzyme, phosphoenol-pyruvate carboxylase, is theprimary carboxylation reaction. The first stable carbon compound is a4-carbon acid which is subsequently decarboxylated. The CO₂ thusreleased is refixed by the C₃ cycle.

[0030] Both C₄ and C₃ plants exhibit a range of ¹³C/¹²C isotopic ratios,but typical values are ca. −10 to −14 per mil (C₄) and −21 to −26 permil (C₃) [Weber et al., J. Agric. Food Chem., 45, 2942 (1997)]. Coal andpetroleum fall generally in this latter range. The ¹³C measurement scalewas originally defined by a zero set by pee dee belemnite (PDB)limestone, where values are given in parts per thousand deviations fromthis material. The “δ¹³C”, values are in parts per thousand (per mil),abbreviated ‰, and are calculated as follows (Equation 6):$\begin{matrix}{{\delta^{13}C} \equiv {\frac{\left( {}^{13}{C/^{12}C} \right)_{sample} - \left( {}^{13}{C/^{12}C} \right)_{standard}}{\left( {}^{13}{C/^{12}C} \right)_{standard}} \times 100\%}} & \left( {{Equation}\quad 6} \right)\end{matrix}$

[0031] Since the PDB reference material (RM) has been exhausted, aseries of alternative RMs have been developed in cooperation with theIAEA, USGS, NIST, and other selected international isotope laboratories.Notations for the per mil deviations from PDB is δ¹³C. Measurements aremade on CO₂ by high precision stable ratio mass spectrometry (IRMS) onmolecular ions of masses 44, 45 and 46.

[0032] Biosourced 1,3-propanediol and the resulting polyol andpolypropylene terephthalate polymer may be completely distinguished fromtheir petrochemical derived counterparts on the basis of ¹⁴C (fm) anddual carbon-isotopic fingerprinting, indicating new compositions ofmatter.

[0033] 1,3-Propanediol and polymers derived therefrom have utility inthe production of polyester fibers and the manufacture of polyurethanes.The new monomer and polymer compositions provided by the instantinvention additionally may be distinguished on the basis of dualcarbon-isotopic fingerprinting from those materials derived solely frompetrochemical sources. The ability to distinguish these products isbeneficial in tracking these materials in commerce. For example,polymers comprising both “new” and “old” carbon isotope profiles may bedistinguished from polymers made only of “old” materials. Hence, theinstant materials may be followed in commerce on the basis of theirunique profile and for the purposes of defining competition, and fordetermining shelf life.

[0034] The following terms and definitions may be used forinterpretation of the claims and specification.

[0035] The abbreviation “AMS” refers to accelerator mass spectrometry.

[0036] The abbreviation “IRMS” refers to measurements of CO₂ by highprecision stable isotope ratio mass spectrometry.

[0037] The terms “genetically altered” or “genetically alteredmicroorganism” refer to any microorganism, suitable for use in thepresent invention, which has undergone an alteration of the nativegenetic machinery of the microorganism. Microorganisms may begenetically altered by undergoing transformation by vectors comprisingheterologous nucleic acid fragments, mutagenesis with mutagenizingagents (e.g., UV light, ethanesulfonic acid) or any other method wherebystable alterations of the cell genome occur.

[0038] The term “construct” refers to a plasmid, virus, autonomouslyreplicating sequence, genome integrating sequence, phage or nucleotidesequence, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell.

[0039] The term “transformation” or “transfection” refers to theacquisition of new genes in a cell after the incorporation of nucleicacid. The acquired genes may be integrated into chromosomal DNA orintroduced as extrachromosomal replicating sequences. The term“transformant” refers to the product of a transformation.

[0040] The term “expression” refers to the transcription and translationto gene product from a gene coding for the sequence of the gene product.

[0041] The term “plasmid” or “vector” or “cosmid” as used herein refersto an extra chromosomal element often carrying genes which are not partof the central metabolism of the cell, and usually in the form ofcircular double-stranded DNA molecules.

[0042] The term “dehydratase enzyme” will refer to any enzyme that iscapable of isomerizing or converting a glycerol molecule to the product3-hydroxypropional. For the purposes of the present invention thedehydratase enzymes include a glycerol dehydratase and a dioldehydratase having preferred substrates of glycerol and 1,2-propanediol,respectively.

[0043] The term “carbon substrate” or “carbon source” means any carbonsource capable of being metabolized by a microorganism wherein thesubstrate contains at least one carbon atom, provided that the carbonsubstrate is other than glycerol or dihydroxyacetone.

[0044] The term “biosourced” means a material derived from a biologicalprocess as opposed to a synthetic, chemical process. “Biosourced”1,3-propanediol is derived from a fermentation process from afermentable carbon source. “Biosourced” polymer or polypropyleneterephthalate refers to polymer comprised in whole or in part frombiosourced monomer.

[0045] The term “polymeric unit” “or repeating unit” means any moleculeor combination of molecules that form a polymeric repeating unit. Forexample, the polymer polypropylene terephthalate is comprised of arepeating unit consisting of 1,3-propanediol and terephthalic acid asshown by formula I:

[0046] Similarly, the polyol derived from 1,3-propanediol, would have arepeating unit of only 1,3-propanediol.

[0047] The term “copolymer” refers to products made by combiningrepeating units of two or more polymers.

[0048] The term “fraction of modern (f_(M))” is defined by NationalInstitute of Standards and Technology (NIST) Standard ReferenceMaterials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxIand HOxII, respectively. The fundamental definition is 0.95 times the¹⁴C/¹²C isotope ratio HOxI, corrected fro radioactive decay since AD1950 and adjusted to a δ¹ ³ C(PDB) reference value of 19.00 permil. Thisis roughly equivalent to decay-corrected pre-Industrial Revolution wood.

[0049] The term “biogeochemical constraints” refers to the minimum andmaximum values for ¹³C and f_(M) ¹⁴C likely to occur in nature. Thesevalues are derived from statistical reasoning (taking into accountfactors that bear on measurement errors, population variations, andeffects of long term meterological trends) as consistent with scientificreasoning based on the pertinent literature of biology, physics,geology, and chemistry. Specifically, the f_(M) ¹⁴C bounds are derivedfrom the observed decay of the nuclear testing pulse in the atmosphere(and biosphere) and from setting the year of growth window to “1990 topresent”. Thus, the range from the f_(M) ¹⁴C bounds for bio-sourced1,3-propanediol are 1.04 to 1.18; and the range for the f_(M) ¹⁴C boundsfor bio-sourced polypropylene terephthalate are 0.28 to 0.32. The ¹³Cbounds additionally take into account a review of the stable isotopeliterature, particularly Fritz and Fontes, Ch. 9, P. Fritz and J. Ch.Fontes, eds., Handbook of Environmental Isotope Geochemistry, Elseview,Amsterdam, 1980, vol. 1, chapter 9. Thus, the range for the δ¹³C for forbio-sourced 1,3-propanediol are −10.9 to −15.4; and the range for δ¹³Cbounds for bio-sourced are polypropylene terephthalate.

[0050] Construction of Recombinant Organisms:

[0051] Recombinant organisms containing the necessary genes that willencode the enzymatic pathway for the conversion of a carbon substrate to1,3-propanediol may be constructed using techniques well known in theart. In the present invention genes encoding dehydratase enzyme wereisolated from a native host such as Klebsiella and used to transform theE. coli host strains DH5α, ECL707 and AA200.

[0052] Methods of obtaining desired genes from a bacterial genome arecommon and well known in the art of molecular biology. For example, ifthe sequence of the gene is known, suitable genomic libraries may becreated by restriction endonuclease digestion and may be screened withprobes complementary to the desired gene sequence. Once the sequence isisolated, the DNA may be amplified using standard primer directedamplification methods such as polymerase chain reaction (U.S. Pat. No.4,683,202) to obtain amounts of DNA suitable for transformation usingappropriate vectors.

[0053] Alternatively, cosmid libraries may be created where largesegments of genomic DNA (35-45 kb) may be packaged into vectors and usedto transform appropriate hosts. Cosmid vectors are unique in being ableto accommodate large quantities of DNA. Generally cosmid vectors have atleast one copy of the cos DNA sequence which is needed for packaging andsubsequent circularization of the foreign DNA. In addition to the cossequence these vectors will also contain an origin of replication suchas Co1E1 and drug resistance markers such as a gene resistant toampicillin or neomycin. Methods of using cosmid vectors for thetransformation of suitable bacterial hosts are well described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, SecondEdition (1989) Cold Spring Harbor Laboratory Press, herein incorporatedby reference.

[0054] Typically to clone cosmids, foreign DNA is isolated and ligated,using the appropriate restriction endonucleases, adjacent to the cosregion of the cosmid vector. Cosmid vectors containing the linearizedforeign DNA is then reacted with a DNA packaging vehicle such asbacteriophage λ. During the packaging process the cos sites are cleavedand the foreign DNA is packaged into the head portion of the bacterialviral particle. These particles are then used to transfect suitable hostcells such as E. coli. Once injected into the cell, the foreign DNAcircularizes under the influence of the cos sticky ends. In this mannerlarge segments of foreign DNA can be introduced and expressed inrecombinant host cells.

[0055] Cosmid vectors and cosmid transformation methods were used withinthe context of the present invention to clone large segments of genomicDNA from bacterial genera known to possess genes capable of processingglycerol to 1,3-propanediol. Specifically, genomic DNA from K.pneumoniae was isolated by methods well known in the art and digestedwith the restriction enzyme Sau3A for insertion into a cosmid vectorSupercos 1™ and packaged using GigapackII packaging extracts. Followingconstruction of the vector E. coli XL1-Blue MR cells were transformedwith the cosmid DNA. Transformants were screened for the ability toconvert glycerol to 1,3-propanediol by growing the cells in the presenceof glycerol and analyzing the media for 1,3-propanediol formation.

[0056] Two of the 1,3-propanediol positive transformants were analyzedand the cosmids were named pKP1 and pKP2. DNA sequencing revealedextensive homology to the glycerol dehydratase gene from C. freundii,demonstrating that these transformants contained DNA encoding theglycerol dehydratase gene. Other 1,3-propanediol positive transformantswere analyzed and the cosmids were named pKP4 and pKP5. DNA sequencingrevealed that these cosmids carried DNA encoding a diol dehydratasegene.

[0057] Although the instant invention utilizes the isolated genes fromwithin a Klebsiella cosmid, alternate sources of dehydratase genesinclude, but are not limited to, Citrobacter, Clostridia, andSalmonella.

[0058] Other genes that will positively affect the production of1,3-propanediol may be expressed in suitable hosts. For example it maybe highly desirable to over-express certain enzymes in the glyceroldegradation pathway and/or other pathways at levels far higher thancurrently found in wild-type cells. This may be accomplished by theselective cloning of the genes encoding those enzymes into multicopyplasmids or placing those genes under a strong inducible or constitutivepromoter. Methods for over-expressing desired proteins are common andwell known in the art of molecular biology and examples may be found inSambrook, supra. Furthermore, specific deletion of certain genes bymethods known to those skilled in the art will positively affect theproduction of 1,3-propanediol. Examples of such methods can be found inMethods in Enzymology, Volume 217, R. Wu editor, Academic Press:SanDiego 1993.

[0059] Mutations and transformations in the 1,3-propanediol productionpathway:

[0060] Representative enzyme pathway. The production of 1,3-propanediolfrom glucose can be accomplished by the following series of steps. Thisseries is representative of a number of pathways known to those skilledin the art. Glucose is converted in a series of steps by enzymes of theglycolytic pathway to dihydroxyacetone phosphate (DHAP) and3-phosphoglyceraldehyde (3-PG). Glycerol is then formed by eitherhydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction, orreduction of DHAP to glycerol 3-phosphate (G3P) followed by hydrolysis.The hydrolysis step can be catalyzed by any number of cellularphosphatases which are known to be non-specific with respect to theirsubstrates or the activity can be introduced into the host byrecombination. The reduction step can be catalyzed by a NAD⁺ (or NADP⁺)linked host enzyme or the activity can be introduced into the host byrecombination. It is notable that the dha regulon contains a glyceroldehydrogenase (E.C. 1.1.1.6) which catalyzes the reversible reaction ofEquation 3.

Glycerol→3-HP+H₂O  (Equation 1)

3-HP+NADH+H⁺→1,3-Propanediol+NAD⁺  (Equation 2)

Glycerol+NAD⁺→DHA+NADH+H⁺  (Equation 3)

[0061] Glycerol is converted to 1,3-propanediol via the intermediate3-hydroxypropionaldehye (3-HP) as has been described in detail above.The intermediate 3-HP is produced from glycerol, Equation 1, by adehydratase enzyme which can be encoded by the host or can introducedinto the host by recombination. This dehydratase can be glyceroldehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28) or anyother enzyme able to catalyze this transformation. Glycerol dehydratase,but not diol dehydratase, is encoded by the dha regulon. 1,3-Propanediolis produced from 3-HP, Equation 2, by a NAD⁺- (or NADP⁺-) linked hostenzyme or the activity can introduced into the host by recombination.This final reaction in the production of 1,3-propanediol can becatalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or otheralcohol dehydrogenases.

[0062] Mutations and transformations that affect carbon channeling. Avariety of mutant organisms comprising variations in the 1,3-propanediolproduction pathway will be useful in the present invention. For examplethe introduction of a triosephosphate isomerase mutation (tpi-) into themicroorganism of the present invention is an example of the use of amutation to improve the performance by carbon channeling. The mutationcan be directed toward a structural gene so as to impair or improve theactivity of an enzymatic activity or can be directed toward a regulatorygene so as to modulate the expression level of an enzymatic activity.

[0063] Alternatively, transformations and mutations can be combined soas to control particular enzyme activities for the enhancement of1,3-propanediol production. Thus it is within the scope of the presentinvention to anticipate modifications of a whole cell catalyst whichlead to an increased production of 1,3-propanediol.

[0064] Media and Carbon Substrates:

[0065] Fermentation media in the present invention must contain suitablecarbon substrates. Suitable substrates may include but are not limitedto monosaccharides such as glucose and fructose, oligosaccharides suchas lactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated.Glycerol production from single carbon sources (e.g., methanol,formaldehyde or formate) has been reported in methylotrophic yeasts (K.Yamada et al., Agric. Biol. Chem., 53:541-543, (1989)) and in bacteria(Hunter et.al., Biochemistry, 24:4148-4155, (1985)). These organisms canassimilate single carbon compounds, ranging in oxidation state frommethane to formate, and produce glycerol. The pathway of carbonassimilation can be through ribulose monophosphate, through serine, orthrough xylulose-monophosphate (Gottschalk, Bacterial Metabolism, SecondEdition, Springer-Verlag: New York (1986)). The ribulose monophosphatepathway involves the condensation of formate with ribulose-5-phosphateto form a 6 carbon sugar that becomes fructose and eventually the threecarbon product glyceraldehyde-3-phosphate. Likewise, the serine pathwayassimilates the one-carbon compound into the glycolytic pathway viamethylenetetrahydrofolate.

[0066] In addition to one and two carbon substrates methylotrophicorganisms are also known to utilize a number of other carbon containingcompounds such as methylamine, glucosamine and a variety of amino acidsfor metabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7th (1993),415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher:Intercept, Andover, UK). Similarly, various species of Candida willmetabolize alanine or oleic acid (Sulter et al., Arch. Microbiol.153:485-489 (1990)). Hence it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety of carboncontaining substrates and will only be limited by the choice oforganism.

[0067] Although it is contemplated that all of the above mentionedcarbon substrates and mixtures thereof are suitable in the presentinvention, preferred carbon substrates are glucose, fructose, sucrose ormethanol.

[0068] In addition to an appropriate carbon source, fermentation mediamust contain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary for1,3-propanediol production. Particular attention is given to Co(II)salts and/or vitamin B₁₂ or precursors thereof.

[0069] Culture Conditions:

[0070] Typically cells are grown at 30° C. in appropriate media.Preferred growth media in the present invention are common commerciallyprepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD)broth or Yeast medium (YM) broth. Other defined or synthetic growthmedia may also be used and the appropriate medium for growth of theparticular microorganism will be known by someone skilled in the art ofmicrobiology or fermentation science. The use of agents known tomodulate catabolite repression directly or indirectly, e.g., cyclicadenosine 2′:3′-monophosphate, may also be incorporated into thereaction media. Similarly, the use of agents known to modulate enzymaticactivities (e.g., methyl viologen) that lead to enhancement of1,3-propanediol production may be used in conjunction with or as analternative to genetic manipulations.

[0071] Suitable pH ranges for the fermentation are between pH 5.0 to pH9.0 where pH 6.0 to pH 8.0 is preferred as the initial condition.

[0072] Reactions may be performed under aerobic or anaerobic conditionswhere anaerobic or microaerobic conditions are preferred.

[0073] Batch and Continuous Fermentations:

[0074] The present process employs a batch method of fermentation. Aclassical batch fermentation is a closed system where the composition ofthe media is set at the beginning of the fermentation and not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the media is inoculated with the desired organism ororganisms and fermentation is permitted to occur adding nothing to thesystem. Typically, however, a “batch” fermentation is batch with respectto the addition of carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. In batchsystems the metabolite and biomass compositions of the system changeconstantly up to the time the fermentation is stopped. Within batchcultures cells moderate through a static lag phase to a high growth logphase and finally to a stationary phase where growth rate is diminishedor halted. If untreated, cells in the stationary phase will eventuallydie. Cells in log phase generally are responsible for the bulk ofproduction of end product or intermediate.

[0075] A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, MukundV., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated byreference.

[0076] Although the present invention is performed in batch mode it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation media is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

[0077] Continuous fermentation allows for the modulation of one factoror any number of factors that affect cell growth or end productconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen level at a fixed rate and allowall other parameters to moderate. In other systems a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the fermentation. Methods of modulating nutrients andgrowth factors for continuous fermentation processes as well astechniques for maximizing the rate of product formation are well knownin the art of industrial microbiology and a variety of methods aredetailed by Brock, supra.

[0078] It is contemplated that the present invention may be practicedusing either batch, fed-batch or continuous processes and that any knownmode of fermentation would be suitable. Additionally, it is contemplatedthat cells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for 1,3-propanediol production.

[0079] Production of 1,3-propanediol from Glycerol

[0080] 1,3-Propanediol produced from glycerol is analyzed in the instantinvention in order to distinguish it from 1,3-propanediol produced fromglucose and other feedstocks.

[0081] 1,3-Propanediol may be generated from glycerol synthetically viaa process that involves (i) dehydration of glycerol over a solidcatalyst, (ii) hydration of acrolein that is produced in (i), andcatalytic hydrogenation of the reaction mixture, containing3-hydroxypropionaldehyde and hydroxyacetone, of stage (ii) [see U.S.Pat. No. 5,426,249, hereby incorporated by reference.] Similarly,1,3-propanediol may be produced by from a reaction mixture of glyceroland synthesis gas in a basic organic solvent in the presence of atungsten and Group VIII metal-containing catalyst composition (see forexample U.S. Pat. No. 4,642,394, hereby incorporated by reference).

[0082] 1,3-Propanediol may also be produced by biological fermentations.For example, Clostridium sp has been used to ferment glycerol to1,3-propanediol under standard anaerobic conditions (U.S. Pat. No.5,254,467) and Citrobacter sp. Have been used for the same purpose undersimilar conditions (U.S. Pat. No. 5,164,309).

[0083] Identification and Purification of 1,3-propanediol:

[0084] Methods for the purification of 1,3-propanediol from fermentationmedia are known in the art. For example propanediols can be obtainedfrom cell media by subjecting the reaction mixture to extraction with anorganic solvent, distillation and column chromatography (U.S. Pat. No.5,356,812). A particularly good organic solvent for this process iscyclohexane (U.S. Pat. No. 5,008,473).

[0085] 1,3-Propanediol may be identified directly by submitting themedia to high pressure liquid chromatography (HPLC) analysis. Preferredin the present invention is a method where fermentation media isanalyzed on an analytical ion exchange column using a mobile phase of0.01N sulfuric acid in an isocratic fashion.

[0086] For industrial applications, purification of 1,3-propanediol fromlarge volumes of fermentor broth requires non-laboratory scale methods.Difficulties to be overcome include removal of cell matter form thebroth (clarification), concentration of 1,3-propanediol either byextraction or water removal and separation of residual impurities fromthe partially purified monomer.

[0087] Broth clarification will typically proceed either by filtration,centrifugation or crossflow microfiltration. Suitable filters aremanufactured for example by Millipore (Millipore Corporation, 80 AshbyRoad, Bedford, Mass.) or Filmtec (Dow Chemical Co.). Centrifugationeffectively removes the bulk of the cells, but, depending upon thenature of the broth, does not always achieve complete cell removal.Crossflow microfiltration yields extremely clear filtrate. Theconcentrate is a slurry rather than a high-solids cake. The skilledperson will be able to adapt the clarification method most appropriatefor the fermentation apparatus and conditions being employed.

[0088] Water reduction of the clarified broth is complicated by the highsolubility of 1,3-propanediol in water. Extraction of 1,3-propanediolfrom the clarified broth may be accomplished by a variety of methods,including evaporation/distillation, membrane technology, extraction byorganic solvent and adsorption.

[0089] Rotary evaporators may be used to initially reduce water volumein the clarified broth. This method has enjoyed good success inApplicants' hands. Precipitation of extraneous proteins and salts do notappear to affect 1,3-propanediol recovery

[0090] Membrane technology may be used either separately or inconjunction with evaporation. Suitable membranes will either (i) allowpassage of 1,3-propanediol, retaining water and other feed molecules(ii) allow passage of water and other molecules, retaining1,3-propanediol or (iii) allow passage of water and 1,3-propanediolwhile retaining other molecules. In the present invention method (iii)is preferred. Particularly useful, are reverse osmosis membranes such asSW-30-2540 (Filmtec, Dow Chemical Co.) and the DL and SH series ofreverse osmosis membranes made by Millipore (Millipore Corporation,Bedford, Mass.).

[0091] Following evaporation and membrane concentration, partiallypurified 1,3-propanediol may be extracted into organic solvents.Suitable solvent will include alcohols such as tert-amyl alcohol,cyciopentanol, octanol, propanol, methanol, and ethanol. Non alcoholsmay also be used such as octanone, cyclohexane and valeraldehyde. Withinthe context of the present invention, alcohols are preferred and ethanolis most preferred.

[0092] Alternatively 1,3-propanediol may be further concentrated byadsorption to various industrial adsorbents. Activated carbon andpolycyclodextrin such as those produced by the American Maize ProductsCompany are particularly suitable.

[0093] Following either extraction or adsorption, partially purified1,3-propanediol must be refined. Refining may be accomplished byelectrodialysis (particularly useful for desalting) which utilizes acombination of anion and cation exchange membranes or biopolar (anionand cation) membranes (see for example, Grandison, Alistair S., Sep.Processes Food Biotechnol. Ind. (1996), 155-177.)

[0094] A preferred method of refining in the present invention isdistillation. Distillation may be done in batch where the operatingpressure is ambient or below, e.g. about 25 in. Hg of vacuum. Monitoringof distillation indicated that materials evaporated in the order offirst to last beginning with light organics, water, diols including1,3-propanediol and finally heavy materials such as glycerol andprecipitated solids.

[0095] Cells:

[0096] Cells suitable in the present invention comprise those thatharbor a dehydratase enzyme. Typically the enzyme will be either aglycerol dehydratase or a diol dehydratase having a substratespecificity for either glycerol or 1,2-propanediol, respectively.Dehydratase enzymes are capable of converting glycerol tohydroxypropionaldehyde (3-HPA) which is then converted to1,3-propanediol. Cells containing this pathway may include mutated orrecombinant organisms belonging to the genera Citrobacter, Enterobacter,Clostridium, Klebsiella, Samonella, and Lactobacillus. Microorganismsknown by persons skilled in the art to produce glycerol by fermentation,e.g., Aspergillus, Saccharomyces, Zygosaccharomyces, Pichia,Kluyveromyces, Candida, Hansenula, Dunaliella, Debaryomyces, Mucor,Torylopsis, and Methylobacteria, may be the hosts for a recombinantdehydratase enzyme. Other cells suitable as hosts in the presentinvention include Bacillus, Escherichia, Pseudomonas and Streptomyces.While not wishing to be bound by theory, it is believed that organisms,belonging to the above mentioned groups, exist in nature that aresuitable for the present invention.

[0097] On the basis of Applicants' experimental work it is contemplatedthat a wide variety of cells may be used in the present invention.Applicants have demonstrated for example that cells varying widely ingenetic and phenotypic composition are able to bioconvert a suitablecarbon substrate to 1,3-propanediol. Cells exemplified include: a K.pneumoniae mutant strain constitutive for the dha genes, recombinant E.coli strains comprising elements of the Klebsiella genome containinggenes encoding either glycerol or diol dehydratase, and recombinant E.coli (tpi⁻) strains also transfected with elements of the Klebsiellagenomes and harboring a mutation in the gene encoding thetriosephosphate isomerase enzyme.

[0098] Although E. coli transformants containing the dha regulon fromKlebsiella pneumonia were able to convert glycerol to 1,3-propanedioleven in the presence of glucose or xylose (Tong et al., Appl. Biochem.Biotech., 34:149 (1992)) no 1,3-propanediol was detected by theseorganisms in the presence of glucose alone. In direct contrast to thisdisclosure, Applicants have discovered that three strains of E. coli,containing either of two independently isolated cosmids comprising thedha regulon from Klebsiella pneumonia, produced 1,3-propanediol from afeed of glucose with no exogenously added glycerol present. E. colistrain ECL707, containing cosmid vectors pKP-1 or pKP-2 comprising theK. pneumoniae dha regulon, showed detectable though modest production of1,3-propanediol from glucose in the absence of exogenously addedglycerol. Recombinant E. coli strains constructed from an alternate hostorganism, DH5α, also containing cosmid vectors pKP-1 or pKP-2, werefound to be more effective than the ECL707 recombinants in producing1,3-propanediol from glucose under the appropriate conditions. Mosteffective in producing 1,3-propanediol from glucose were the recombinantE. coli strains AA200 containing cosmid vectors pKP-1 or pKP-2. E. colistrain AA200 contains a defective triosephosphate isomerase enzyme(tpi⁻).

[0099] A strain of AA200-pKP1, selected for further study from a pool ofindependent isolates from the transformation reaction, converted glucoseto 1,3-propanediol in a two stage reaction. In the first stage, thestrain AA200-pKP1-5 was grown to high cell density in the absence ofglucose and glycerol. In the second stage, the grown cells, suspended ina medium containing glucose but no glycerol, converted glucose to1,3-propanediol with high conversion and selectivity. Although differingimmumochemically, chromato-graphically, and genetically, the coenzymeB₁₂-dependent enzymes glycerol dehydratase (E.C. 4.2.1.30) and dioldehydratase (E.C. 4.2.1.28) catalyze the conversion of glycerol to3-hydroxypropionaldehyde. Glycerol dehydratase, but not dioldehydratase, is encompassed by the dha regulon. K. pneumoniae ATCC 8724,containing a diol dehydratase but not a glycerol dehydratase convertsglycerol to 1,3-propanediol (Forage et al., J. Bacteriol., 149:413,(1982)). Recombinant E. coli strains ECL707 and AA200, containing cosmidvector pKP4 encoding genes for a diol dehydratase, converted glucose to1,3-propanediol.

[0100]K. pneumoniae ECL2106, prepared by mutagenesis from a naturallyoccurring strain (Ruch et al., J. Bacteriol. 124:348 (1975)), exhibitsconstitutive expression of the dha regulon (Ruch et al., supra; Johnsonet al., J. Bacteriol. 164:479 (1985)). A strain derived from K.pneumoniae ATCC 25955, displaying the same phenotype, has been similarlyprepared (Forage et al., J. Bacteriol. 149:413 (1982)). Expression ofthe Klebsiella dha structural genes is, in part, controlled by arepressor (product of dha R) (Sprenger et al., J. Gen Microbiol.135:1255 (1989)). Applicants have shown that ECL2106, which isconstitutive for the dha structural genes, produced 1,3-propanediol froma feed of glucose in the absence of exogenously added glycerol, Example5. This is in contrast to wild type K. pneumoniae ATCC 25955 which didnot produce detectable levels of 1,3-propanediol under the sameconditions, Example 5.

[0101] The expression of the dha structural genes in ECL2106 is furthercontrolled by catabolite expression (Sprenger et al., J. Gen Microbiol.135:1255 (1989)). Elimination of catabolite repression can be achievedby placing the necessary structural genes under the control of alternatepromoters as has been demonstrated for 1,3-propanediol oxidoreductase(dhaT) from C. freundii and diol dehydratase from K. oxytoca ATCC 8724(Daniel et al., J. Bacteriol. 177:2151 (1995) and Tobimatsu et al., J.Biol. Chem. 270:7142 (1995)). By eliminating catabolite repression fromECL2106 in this manner, an improvement in the production of1,3-propanediol from glucose in the absence of an exogenous source ofglycerol is achieved. An even further improvement is obtained byappropriate carbon channeling as is described, by example, with the tpi⁻mutation.

[0102] As the dha regulons of Citrobacter and Klebsiella sp. arestrikingly similar, one of skill in the art will appreciate thatteachings that involve the production of 1,3-propanediol from glucose inthe absence of an exogenous source of glycerol for Klebsiella sp.applies to Citrobacter sp. as well. Furthermore, as the metabolism ofglycerol by C. butyricum is comparable to that of K. pneumoniae (Zeng etal., Biotechnol. and Bioeng. 44:902 (1994)), teachings will extend toClostridia sp. as well.

[0103] Sample Preparation Prior to Isotopic Analysis and IsotopicMeasurements

[0104] Samples subjected to analysis by ¹³C and ¹⁴C dual isotopiccharacterization first underwent quantitative combustion of carbon tocarbon dioxide. Analysis was accomplished by one of 2 methods, “closedtube” or via commercial “CHN” analyzer. The closed type method involvedheating the sample in the presence of CuO, as an oxygen source in aclosed tube. The commercial analyzer used molecular oxygen as a oxygensource. Evolving CO₂ was purified and submitted for analysis acceleratormass spectrometry (AMS) and isotope ratio mass spectrometry (IRMS).

[0105]¹⁴C was determined by AMS, using “conventional” graphite targetsprepared from the purified CO₂. Oxalic acid isotope standards were usedfor standardization. ¹³C was determined on a split of the purified CO₂samples using an “Optima” isotope ratio mass spectrometer, and the“Craig” algorithm (Allison et al., Proceedings of a Consultants' Meetingon Reference and intercomparison materials for stable isotopes of lightelements (1993), pp 155-162) operating on the mass 44, 45 and 46currents. The index used for ¹³C wasδ¹³C=[(¹³C/¹²C)_(sample)−(¹³C/¹²C)_(standard)/(¹³C/¹²C)_(standard)×1000‰[Weber et al., J. Agric. Food Chem. 45, 2942, (1997)]. The index usedfor ¹⁴C was fraction of modern (f_(M))¹⁴C.

[0106] Based on this analysis 1,3-propanediol derived from glucose wasfound to have a δ¹³C of about −13.84% to about −13.92%, and a f_(M) ¹⁴Cof about 1.11 to about 1.124. 1,3-Propanediol derived from glycerol wasfound to have a δ¹³C of about −22.41% to about −22.60%, and a f_(M) ¹⁴Cof about 0.85 to about 0.89. In contrast 1,3-propanediol derived frompetrochemical sources (acrolein) was found to have a δ¹³C of about−17.95% to about −18.33%, and a f_(M) ¹⁴C of about −0.004 to about0.007. Polypropylene terephthalate derived from glucose was found tohave a δ¹³C of about −24.74% to about −24.88%, and a f_(M) ¹⁴C of about0.299 to about 0.309.

[0107] The present invention is further defined in the followingExamples. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

EXAMPLES

[0108] GENERAL METHODS

[0109] Procedures for phosphorylations, ligations and transformationsare well known in the art. Techniques suitable for use in the followingexamples may be found in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989).

[0110] Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found in Manual of Methods forGeneral Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds), American Society for Microbiology, Washington, D.C.(1994) or Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass. All reagents and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

[0111] Glycerol used in the production of 1,3-propanediol was obtainedfrom J. T. Baker Glycerin USP grade, Lot J25608 and G19657.

[0112] NBS19 was used as the standard for ¹³C (see Allison et al.,Proceedings of a Consultants' Meeting on Reference and intercomparisonmaterials for stable isotopes of light elements (1993), pp 155-162).

[0113] NIST SRMs 4990B (HOxI) and 4990C (HOxII) were used as standardsfor ¹⁴C.

[0114] The meaning of abbreviations is as follows: “h” means hour(s),“min” means minute(s), “sec” means second(s), “d” means day(s), “mL”means milliliters, “L” means liters, 50amp is 50 μg/mL ampicillin, andLB-50amp is Luria-Bertani broth containing 50 μg/mL ampicillin.

[0115] Within the tables the following abbreviations are used. “Con.” isconversion, “Sel.” is selectivity based on carbon, and “nd” is notdetected.

[0116] Enzyme Assays:

[0117] Glycerol dehydratase activity in cell free extracts wasdetermined using 1,2-propanediol as substrate. The assay, based on thereaction of aldehydes with methylbenzo-2-thiazolone hydrazone, has beendescribed (Forage and Foster, Biochim. Biophys. Acta, 569:249 (1979)).The activity of 1,3-propanediol oxidoreductase, sometimes referred to as1,3-propanediol dehydrogenase, was determined in solution or in slabgels using 1,3-propanediol and NAD⁺ as substrates as has also beendescribed. Johnson and Lin, J. Bacteriol., 169:2050 (1987).

[0118] Isolation and Identification 1,3-Propanediol:

[0119] The conversion of glycerol to 1,3-propanediol was monitored byHPLC. Analyses were performed using standard techniques and materialsavailable to one of skill in the art of chromatography. One suitablemethod utilized a Waters Maxima 820 HPLC system using UV (210 nm) and RIdetection. Samples were injected onto a Shodex SH-1011 column (8 mm×300mm, purchased from Waters, Milford, Mass.) equipped with a ShodexSH-1011P precolumn (6 mm×50 mm), temperature controlled at 50° C., using0.01 N H₂SO₄ as mobile phase at a flow rate of 0.5 mL/min. Whenquantitative analysis was desired, samples were prepared with a knownamount of trimethylacetic acid as external standard. Typically, theretention times of glycerol (RI detection), 1,3-propanediol (RIdetection), and trimethylacetic acid (UV and RI detection) were 20.67min, 26.08 min, and 35.03 min, respectively.

[0120] Production of 1,3-propanediol was confirmed by GC/MS. Analyseswere performed using standard techniques and materials available to oneof skill in the art of GC/MS. One suitable method utilized a HewlettPackard 5890 Series II gas chromatograph coupled to a Hewlett Packard5971 Series mass selective detector (EI) and a HP-INNOWax column (30 mlength, 0.25 mm i.d., 0.25 micron film thickness). The retention timeand mass spectrum of 1,3-propanediol generated from glycerol werecompared to that of authentic 1,3-propanediol (m/e: 57, 58).

[0121] Construction of K. pneumoniae Cosmid Libraries:

[0122]K. pneumoniae (ATCC 25955) was grown in 100 ml LB medium for 8 hat 37° C. with aeration. Bacteria (25 mL per tube) were centrifuged at3,000 rpm for 15 min in a DuPont Sorvall GLC 2.B centrifuge at roomtemperature. The bacteria were pelleted and supernatant was decanted.The bacterial cell pellet was frozen at −20° C. The chromosomal DNA wasisolated as outlined below with special care taken to avoid shearing ofDNA (i.e., vortexing was avoided). One tube of bacteria was resuspendedin 2.5 mL of 50 mM Tris-10 mM EDTA and 500 μL of lysozyme (1 mg/mL) wasadded. The pellet was gently resuspended and the suspension wasincubated at 37° C. for 15 min. Sodium dodecyl sulfate was added tobring the final concentration to 0.5%. This resulted in the solutionbecoming clear. Proteinase K (50 μg/mL) was added and the suspension wasincubated at 55° C. for 2 h. The tube was removed and transferred to anice bath and sodium chloride was added to yield a 0.4 M finalconcentration. Two volumes of ethanol were added to the liquid. A glasstube was inserted to the interface and the DNA was gently spooled. DNAwas dipped into a tube containing 70% ethanol. After drying in vacuo,the DNA was resuspended in 500 μL of water and the concentration of DNAwas determined spectrophoto-metrically. A diluted aliquot of DNA was runon a 0.5% agarose gel to determine the intact nature of DNA.

[0123] The chromosomal DNA was partially digested with Sau3A as outlinedby Sambrook et al., supra. DNA (2 μg) was digested with 2 units of Sau3A(Promega, Madison, Wis.) at room temperature in 200 μL of total volume.At 0, 5, 10 and 20 min, samples (50 μL) were removed and transferred totubes containing 5 umol of EDTA. These tubes were incubated at 70° C.for 10 min. An aliquot (2 μL) was withdrawn and analyzed on a 0.5%agarose gel electrophoresis to determine the level of digestion and therest of the sample (48 μL) was stored at −20° C. The gel was stainedwith ethidium bromide and visualized under UV to determine the partialdigestion of the chromosomal DNA. A decrease in the size of thechromosomal DNA with increase in time was observed showing that thedecrease in the size of the chromosomal DNA is due to the action ofSau3A. DNA was extracted from rest of the sample by standard protocolmethods (Sambrook et al., supra).

[0124] A cosmid library of partially digested DNA from K. pneumoniae wasprepared using Supercos cosmid vector kit and GigapackII packagingextracts using reagents purchased from Stratagene (La Jolla, Calif.).The instructions provided by the manufacturer were followed. Thepackaged K. pneumoniae contained 4×10⁴ to 1.0×10⁵ phage titer asdetermined by transfecting E. coli XL1-Blue MR.

[0125] Cosmid DNA was isolated from 6 of the E. coli transformants andfound to contain large insert of DNA (25 to 30 kb).

Example 1

[0126] Cloning and transformation of E. coli host cells with cosmid DNA

for the expression of 1,3-propanediol

[0127] Media

[0128] Synthetic S12 medium was used in the screening of bacterialtransformants for the ability to make 1,3-propanediol. S12 mediumcontains: 10 mM ammonium sulfate, 50 mM potassium phosphate buffer, pH7.0, 2 mM MgCl₂, 0.7 mM CaCl₂, 50 μM MnCl₂, 1 μM FeCl₃, 1 μM ZnCl, 1.7μM CuSO₄, 2.5 μM CoCl₂, 2.4 μM Na₂MoO₄, and 2 μM thiamine hydrochloride.

[0129] Medium A used for growth and fermentation consisted of: 10 mMammonium sulfate; 50 mM MOPS/KOH buffer, pH 7.5; 5 mM potassiumphosphate buffer, pH 7.5; 2 mM MgCl₂; 0.7 mM CaCl₂; 50 μM MnCl₂; 1 μMFeCl₃; 1 μM ZnCl; 1.72 μM CuSO₄; 2.53 μM CoCl₂; 2.42 μM Na₂MoO₄; 2 μMthiamine hydrochloride; 0.01% yeast extract; 0.01% casamino acids; 0.8μg/mL vitamin B₁₂; and 50amp. Medium A was supplemented with either 0.2%glycerol or 0.2% glycerol plus 0.2% D-glucose as required.

[0130] Cells:

[0131]Klebsiella pneumoniae ECL2106 (Ruch et al., J. Bacteriol., 124,348 (1975)), also known in the literature as K. aerogenes or Aerobacteraerogenes, was obtained from E. C. C. Lin (Harvard Medical School,Cambridge, Mass.) and was maintained as a laboratory culture.

[0132]Klebsiella pneumoniae ATCC 25955 was purchased from American TypeCulture Collection (Manassas, Va.).

[0133]E. coli DH5α was purchased from Gibco/BRL and was transformed withthe cosmid DNA isolated from Klebsiella pneumoniae ATCC 25955 containinga gene coding for either a glycerol or diol dehydratase enzyme. Cosmidscontaining the glycerol dehydratase were identified as pKP1 and pKP2 andcosmid containing the diol dehydratase enzyme were identified as pKP4.Transformed DH5α cells were identified as DH5α-pKP1, DH5α-pKP2, andDH5α-pKP4.

[0134]E. coli ECL707 (Sprenger et al., J. Gen. Microbiol., 135, 1255(1989)) was obtained from E. C. C. Lin (Harvard Medical School,Cambridge, Mass.) and was similarly transformed with cosmid DNA fromKlebsiella pneumoniae. These transformants were identified asECL707-pKP1 and ECL707-pKP2, containing the glycerol dehydratase geneand ECL707-pKP4 containing the diol dehydratase gene.

[0135]E. coli AA200 containing a mutation in the tpi gene (Anderson etal., J. Gen Microbiol., 62, 329 (1970)) was purchased from the E. coliGenetic Stock Center, Yale University (New Haven, Conn.) and wastransformed with Klebsiella cosmid DNA to give the recombinant organismsAA200-pKP 1 and AA200-pKP2, containing the glycerol dehydratase gene,and AA200-pKP4, containing the diol dehydratase gene.

[0136] DH5α:

[0137] Six transformation plates containing approximately 1,000 coloniesof E. coli XL1-Blue MR transfected with K. pneumoniae DNA were washedwith 5 mL LB medium and centrifuged. The bacteria were pelleted andresuspended in 5 mL LB medium+glycerol. An aliquot (50 μL) wasinoculated into a 15 mL tube containing S12 synthetic medium with 0.2%glycerol+400 ng per mL of vitamin B₁₂+0.001% yeast extract+50amp. Thetube was filled with the medium to the top and wrapped with parafilm andincubated at 30° C. A slight turbidity was observed after 48 h.Aliquots, analyzed for product distribution as described above at 78 hand 132 h, were positive for 1,3-propanediol, the later time pointscontaining increased amounts of 1,3-propanediol.

[0138] The bacteria, testing positive for 1,3-propanediol production,were serially diluted and plated onto LB-50amp plates in order toisolate single colonies. Forty eight single colonies were isolated andchecked again for the production of 1,3-propanediol. Cosmid DNA wasisolated from 6 independent clones and transformed into E. coli strainDH5α. The transformants were again checked for the production of1,3-propanediol. Two transformants were characterized further anddesignated as DH5α-pKP1 and DH5α-pKP2.

[0139] A 12.1 kb EcoRI-SaII fragment from pKP1, subcloned into pIBI31(IBI Biosystem, New Haven, Conn.), was sequenced and termed pHK28-26(SEQ ID NO:1). Sequencing revealed the loci of the relevant open readingframes of the dha operon encoding glycerol dehydratase and genesnecessary for regulation. Referring to SEQ ID NO:1, a fragment of theopen reading frame for dhaK encoding dihydroxyacetone kinase is found atbases 1-399; the open reading frame dhaD encoding glycerol dehydrogenaseis found at bases 983-2107; the open reading frame dhaR encoding therepressor is found at bases 2209-4134; the open reading frame dhaTencoding 1,3-propanediol oxidoreductase is found at bases 5017-6180; theopen reading frame dhaB1 encoding the alpha subunit glycerol dehydrataseis found at bases 7044-8711; the open reading frame dhaB2 encoding thebeta subunit glycerol dehydratase is found at bases 8724-9308; the openreading frame dhaB3 encoding the gamma subunit glycerol dehydratase isfound at bases 9311-9736; and the open reading frame dhaBX, encoding aprotein of unknown function is found at bases 9749-11572.

[0140] Single colonies of E. coli XL1-Blue MR transfected with packagedcosmid DNA from K. pneumoniae were inoculated into microtiter wellscontaining 200 uL of S15 medium (ammonium sulfate, 10 mM; potassiumphosphate buffer, pH 7.0, 1 mM; MOPS/KOH buffer, pH 7.0, 50 mM; MgCl₂, 2mM; CaCl₂, 0.7 mM; MnCl₂, 50 uM; FeCl₃, 1 uM; ZnCl, 1 uM; CuSO₄, 1.72uM; CoCl₂, 2.53 uM; Na₂MoO₄, 2.42 uM; and thiamine hydrochloride, 2uM)+0.2% glycerol+400 ng/mL of vitamin B₁₂+0.001% yeast extract+50 ug/mLampicillin. In addition to the microtiter wells, a master platecontaining LB-50amp was also inoculated. After 96 h, 100 uL waswithdrawn and centrifuged in a Rainin microfuge tube containing a 0.2micron nylon membrane filter. Bacteria were retained and the filtratewas processed for HPLC analysis. Positive clones demonstrating1,3-propanediol production were identified after screening approximately240 colonies. Three positive clones were identified, two of which hadgrown on LB-50amp and one of which had not. A single colony, isolatedfrom one of the two positive clones grown on LB-50amp and verified forthe production of 1,3-propanediol, was designated as pKP4. Cosmid DNAwas isolated from E. coli strains containing pKP4 and E. coli strainDH5α was transformed. An independent transformant, designated asDH5α-pKP4, was verified for the production of 1,3-propanediol.

[0141] ECL707:

[0142]E. coli strain ECL707 was transformed with cosmid K. pneumoniaeDNA corresponding to pKP1, pKP2, pKP4 and the Supercos vector alone andnamed ECL707-pKP1, ECL707-pKP2, ECL707-pKP4, and ECL707-sc,respectively. ECL707 is defective in glpK, gld, and ptsD which encodethe ATP-dependent glycerol kinase, NAD⁺-linked glycerol dehydrogenase,and enzyme II for dihydroxyacetone of the phosphoenolpyruvate dependentphosphotransferase system, respectively.

[0143] Twenty single colonies of each cosmid transformation and five ofthe Supercos vector alone (negative control) transformation, isolatedfrom LB-50amp plates, were transferred to a master LB-50amp plate. Theseisolates were also tested for their ability to convert glycerol to1,3-propanediol in order to determine if they contained dehydrataseactivity. The transformants were transferred with a sterile toothpick tomicrotiter plates containing 200 μL of Medium A supplemented with either0.2% glycerol or 0.2% glycerol plus 0.2% D-glucose. After incubation for48 hr at 30° C., the contents of the microtiter plate wells werefiltered through an 0.45 μ nylon filter and chromatographed by HPLC. Theresults of these tests are given in Table 1. TABLE 1 Conversion ofglycerol to 1,3-propanediol by transformed ECL707: number of positiveisolates/number of isolates tested Transformant Glycerol Glycerol plusGlucose ECL707-pKP1 19/20 19/20 ECL707-pKP2 18/20 20/20 ECL707-pKP4 0/20 20/20 ECL707-sc 0/5 0/5

[0144] AA200:

[0145]E. coli strain AA200 was transformed with cosmid K. pneumoniae DNAcorresponding to pKP1, pKP2, pKP4 and the Supercos vector alone andnamed AA200-pKP1, AA200-pKP2, AA200-pKP4, and AA200-sc, respectively.Strain AA200 is defective in triosephosphate isomerase, (tpi⁻).

[0146] Twenty single colonies of each cosmid transformation and five ofthe empty vector transformation were isolated and tested for theirability to convert glycerol to 1,3-propanediol as described for E. colistrain ECL707. The results of these tests are given in Table 2. TABLE 2Conversion of glycerol to 1,3-propanediol by transformed AA200: Numberof positive isolates/number of isolates tested Transformant GlycerolGlycerol plus Glucose AA200-pKP1 17/20 17/20 AA200-pKP2 17/20 17/20AA200-pKP4  2/20 16/20 AA200-sc 0/5 0/5

Example 2 Conversion of D-glucose to 1.3-propanediol by E. coli strainAA200, transformed with Klebsiellia pneumoniae DNA containingdehydratase activity

[0147] Glass serum bottles, filled to capacity with media (ca. 14 mL ofMedium A as defined in Example 1 supplemented with 10 μg/mL kanamycinand 0.2% D-glucose, plus or minus 0.5-1.0 mM cyclic adenosine2′:3′-monophosphate (cAMP)), were innoculated with selected singlecolony isolates of E. coli strain AA200 containing the K. pneumoniae dharegulon cosmids pKP1 or pKP2, the K. pneumoniae pdu operon pKP4, or theSupercos vector alone. In order to avoid contact with glycerol, theinnoculation was performed from either an agar plate of LB-50amp or froma liquid culture of the same medium. The reactions were incubated forca. 72 hr at 30° C. while shaking at 250 rpm. Growth was determined bythe change in absorbance at 600 nm where the initial OD₆₀₀ was 0.020 AU.The extent of glucose depletion and product distribution were determinedby HPLC. Single colony isolates are identified by a numbered suffix“−x”, e.g., AA200-pKP1−x. Cumulative results are presented in Table 3and Table 4. TABLE 3 Conversion of 0.2% D-glucose to 1,3-propanediol bytransformed E. coli strain AA200: without cAMP [1,3-propane-Transformant OD₆₀₀ diol] (mM) Con. (%) Sel. (%) AA200-pKP1-3 0.056  0.0517 1 AA200-pKP1-5 0.115 nd  0 ″ 0.007 nd  0 ″ 0.076 0.2 14 5AA200-pKP1-20 0.116 nd 27 0 ″ 0.205 0.3 17 8 AA200-pKP2-10 0.098 0.2 137 AA200-pKP2-14 0.117 0.5 17 14  ″ 0.129 0.2 19 5 AA200-pKP2-20 0.094 nd11 0 AA200-pKP4-4 0.198 0.1 28 2 AA200-pKP4-19 0.197 0.2 34 3AA200-pKP4-20 0.206 0.1 38 1 AA200-sc-1 0.097 nd 22 0 ″ 0.176 nd 46 0

[0148] TABLE 4 Conversion of 0.2% D-glucose to 1,3-propanediol bytransformed E. coli strain AA200: with cAMP [1,3-propane- TransformantOD₆₀₀ diol] (mM) % Con. % Sel. AA200-pKP1-3 0.102 0.5 19 12 AA200-pKP1-50.088 1.5 21 37 ″ 0.236 1.4 24 28 ″ 0.071 0.8 15 23 AA200-pKP1-20 0.153nd 40 0 ″ 0.185 0.9 27 16 AA200-pKP2-10 0.098 0.2 13 7 AA200-pKP2-140.213 2.0 26 27 ″ 0.155 0.6 25 12 AA200-pKP2-20 0.198 1.2 40 14AA200-pKP4-4 0.218 0.1 31 2 AA200-pKP4-19 0.223 0.2 37 3 AA200-pKP4-200.221 0.2 35 3 AA200-sc-1 0.111 nd 23 0 ″ 0.199 nd 49 0 ″ 0.122 nd 25 0

Example 3 Conversion of D-glucose to 1,3-propanediol by E. coli strainDH5α, transformed with Klebsiellia pneumoniae DNA containing dehydrataseactivity

[0149]E. coli strain DH5α, containing the K. pneumoniae dha reguloncosmids pKP1 or pKP2, were tested for their ability to convert D-glucoseto 1,3-propanediol as described in Example 2. The results are presentedin Table 5. TABLE 5 Conversion of 0.2% D-glucose to 1,3-propanediol bytransformed E. coli strain DH5α: plus (+) and minus (−) cAMP[1,3-propane- Transformant OD₆₀₀ diol] (mM) % Con. % Sel. DH5α-pKP1 (−)0.630 0.5 100 2 DH5α-pKP1 (+) 0.774 0.6 100 3 DH5α-pKP2 (−) 0.584 0.6100 3 DH5α-pKP2 (+) 0.699 0.7 100 3

Example 4 Conversion of D-glucose to 1,3-propanediol by E. coli strainECL707, transformed with Klebsiellia pneumoniae DNA containingdehydratase activity

[0150]E. coli strain ECL707, containing the K. pneumoniae dha reguloncosmids pKP1 or pKP2, the K. pneumoniae pdu operon pKP4, or the Supercosvector alone, were tested for their ability to convert D-glucose to1,3-propanediol as described in Example 2. In each case, conversion wasquantitative. The results are presented in Table 6. TABLE 6 Conversionof D-glucose to 1,3-propanediol by transformed E. coli strain ECL707:with and without cAMP [1,3-propane- [1,3-propane- OD₆₀₀ diol] (mM) OD₆₀₀diol] (mM) Transformant (without cAMP) (with cAMP) ECL707-pKP1-1 0.6070.1 0.475 0.1 ECL707-pKP1-3 0.619 0.1 0.422 0.1 ECL707-pKP1-7 0.582 0.20.522 0.2 ECL707-pKP1-10 0.593 0.1 0.408 0.1 ECL707-pKP1-18 0.584 0.10.433 0.1 ECL707-pKP2-4 0.588 0.1 0.408 0.1 ECL707-pKP2-5 0.630 0.10.516 0.2 ECL707-pKP2-8 0.542 0.1 0.486 0.1 ECL707-pKP2-15 0.589 0.10.485 0.1 ECL707-pKP2-19 0.577 0.1 0.504 0.1 ECL707-pKP4-8 0.499 nd0.361 <0.1 ECL707-pKP4-9 0.544 nd 0.354 nd ECL707-pKP4-10 0.515 nd 0.265<0.1 ECL707-pKP4-14 0.512 nd 0.318 <0.1 ECL707-pKP4-17 0.545 nd 0.388<0.1 ECL707-sc-1 0.592 nd 0.385 nd

Example 5 Conversion of D-glucose to 1,3-propanediol under fermentationconditions

[0151]E. coli strain ECL707, containing the K. pneumoniae dha reguloncosmids pKP1 or pKP2, the K. pneumoniae pdu operon pKP4, or the Supercosvector alone, is grown in a 5 L Applikon fermenter for the production of1,3-propanediol from glucose.

[0152] The medium used contains 50-100 mM potassium phosphate buffer, pH7.5, 40 mM (NH₄)₂SO₄, 0.1% (w/v) yeast extract, 10 μM CoCl₂, 6.5 μMCuCl₂, 100 μM FeCl₃, 18 μM FeSO₄, 5 μM H₃BO₃, 50 μM MnCl₂, 0.1 μMNa₂MoO₄, 25 μM ZnCl₂, 0.82 mM MgSO₄, 0.9 mM CaCl₂, and 10-20 g/Lglucose. Additional glucose is fed, with residual glucose maintained inexcess. Temperature is controlled at 37° C. and pH controlled at 7.5with 5N KOH or NaOH. Appropriate antibiotics are included for plasmidmaintenance. For anaerobic fermentations, 0.1 vvm nitrogen is spargedthrough the reactor; when the dO setpoint was 5%, 1 vvm air is spargedthrough the reactor and the medium is supplemented with vitamin B₁₂.

[0153] Titers of 1,3-propanediol (g/L) range from 8.1 to 10.9. Yields of1,3-propandiol (g/g) range from 4% to 17%.

Example 6 Purification of Biosourced 1,3-Propanediol

[0154] 1,3-Propanediol, produced as recited in Examples 2-5, waspurified, by a multistep process including broth clarification, rotaryevaporation, anion exchange and multiple distillation of thesupernatant.

[0155] At the end of the fermentation, the broth was clarified using acombination of centrifugation and membrane filtration for cellseparation, followed by ultrafiltration through a 1000 MW membrane. Theclarified broth processed in a large rotary evaporator. Approximately 46pounds of feed material (21,000 grams) were processed to a concentratedsyrup. A 60 ml portion of syrup was placed in the still pot of a 1″diameter distillation column. Distillation was conducted at a vacuum of25 inches of mercury. A reflux ratio of approximately 1 was usedthroughout the distillation. Several distillate cuts were taken, thecentral of which received further processing. The material was dilutedwith an equal volume of water, the material was loaded onto an anionexchange column (mixed bed, 80 grams of NM-60 resin), which had beenwater-washed. Water was pumped at a rate of 2 ml/min, with fractionsbeing collected every 9 minutes. Odd number fractions were analyzed, andfractions 3 through 9 contained 3G. The fractions containing 3G werecollected and subjected to microdistillation to recover several grams ofpure 1,3-propanediol monomer (which was polymerized to polypropyleneterephthalate according the method described in Example 7.

Example 7 Polymerization of Biosourced 1,3-Propanediol to PolypropyleneTerephthalate

[0156] Dihydroxypropyl terephthalate, purified according to the methodrecited in Example 6 was produced from dimethyl terephthalate as apolypropylene terephthalate monomer according to the following process.

[0157] Dimethyl terephthalate (150 g) and biosourced 1,3-propanediol aremixed together with titanium isopropoxide (registry number 546-68-9)(0.03 mL) in a 1-liter flask equipped with a stirrer, a thermometer anda 13-inch Vigreaux condenser leading to a distillation head. The mixtureis blanketed with nitrogen and heated to react the components anddistill off the methanol reaction by-product. After about 5 h, 61 mL ofmethanol distilled off, close to the stoichiometric quantity expected.This dihydroxy propyl terephthalate is then cast into an aluminum trayand allowed to solidify.

[0158] Preparation of dihydroxypropyl terephthalate from terephthalicacid as a

[0159] polypropylene terephthalate monomer:

[0160] A 100-mL round bottom flask equipped with a stir bar, nitrogensource and a distillation head was charged with terephthalic acid (33.2g) and 1,3-propanediol (30.4 g, Example 2). The reaction was heated withstirring under nitrogen to distill water and 1,3-propanediol from themixture, until no further distillate appeared in the receiving flask,and the terephthalic acid is in solution. This result typically occursafter 20 to 28 h. The pressure was then reduced in the flask by means ofa vacuum pump to distill additional water and 1,3-propanediol as theesterification takes place. The esterification was judged as beingcomplete when the 1,3-propanediol and water cease to distill, typically2 h at 25 mm Hg pressure. The reaction mixture was clear, indicatingthat the terephthalic acid had dissolved and esterified. The moltenterephthalic acid/1,3-propanediol oligomer was then cast from the flaskinto an aluminum pan under nitrogen and allowed to solidify.

[0161] Polymerization of dihydroxypropyl terephthalate to polypropyleneterephthalate:

[0162] A 500-mL round bottom reaction flask equipped with a distillationhead, mechanical stirrer, nitrogen source, and a vacuum source wascharged with 150 g of the dihydroxypropyl terephthalate monomer,prepared as described in either of the two procedures above. Thepolymerization catalyst, titanium isopropoxide (0.022 mL), was thenadded to the reaction flask. The flask was immersed in a molten metalbath equilibrated at 255° C. Stirring at 50 rpm commenced as soon as themonomer melted, and the melt was held at 255° C. and 1 atm of pressurefor 30 min. The pressure was then reduced to 120 mm Hg pressure for 20min, then to 20 mm Hg for 10 min, and then to 10 mm Hg for an additional10 min. Finally, the pressure was reduced to less than 1 mm Hg for theduration of the polymerization, as indicated by a torque rise, whichoccurred after an additional 1 h, yielding higher molecular weightpolypropylene terephthalate.

Example 8 Dual Isotopic Characterization—Distinct ProductCharacterization

[0163] Example 8 demonstrates that biosourced 1,3-propanediol and itspolymer derivative may be distinguished from monomer and polymersderived solely from petrochemical sources.

[0164] Samples analyzed by ¹³C and ¹⁴C dual isotopic characterizationare listed in Table 7 and included glucose (samples 1, 2), polypropyleneterephthalate produced from glucose (samples 3, 4), 1,3-propanediolproduced from glucose (sample 5), 1,3-propanediol produced from glycerol(sample 6, see Example 1, Table 1) and 1,3-propanediol produced frompetrochemical feedstock (samples 7, 8).

[0165] Petrochemical derived 1,3-propanediol was obtained from DegussaAktiengesellschaft (Frankfurt, Federal Republic of Germany) and preparedobtained by hydration of acrolein to 3-hydroxypropionaldehyde withsubsequent catalytic hydrogenation, according to the process describedin U.S. Pat. No. 5,364,987, hereby incorporated by reference.

[0166] Sample Preparation Prior to Isotopic Analysis (combustion):

[0167] The first step comprises quantitative combustion of the samplecarbon to carbon dioxide. Two alternate routes were used for thisoxidation: (1) use of closed tube (CT) combustion, heating the sample to900° C. in a quartz tube with CuO as the oxygen source; (2) using aspecially adapted commercial “CHN” analyzer for a CO₂ trapping system.In the latter case, the oxygen source was molecular (tank) oxygen.Recovery of test materials was evaluated for both systems, based on puresubstance stoichiometry. Sample CO₂ was purified and sealed in quartztubes and submitted for isotopic measurement by AMS and IRMS. As thesesamples were all in the form of CO₂, “memory” of the original chemicalsubstances was totally erased. The amounts of material oxidized rangedfrom ca. 0.6 to 2.0 mg carbon, quantities suitable for high precisionmeasurement.

[0168] Isotopic Measurements:

[0169]¹⁴C was determined by AMS, using “conventional” graphite targetsprepared from the CO₂. The accelerator employed was the NSF AMSfacility, based on 2 MV tandem AMS with monitoring of C³⁺ atomic ¹³C and¹⁴C high energy ions. Both HOxI and HoxII standards were used, affordingthe opportunity to check the precision and bias of the process bymonitoring the HOxII/HOxI ratio. Typically for AMS measurements at themg level, this measured ratio is stable to ca. 1%.

[0170]¹³C was determined on a split of the CO₂ samples using an “Optima”isotope ratio mass spectrometer, and the “Craig” algorithm (Allison etal., Proceedings of a Consultants' Meeting on Reference andintercomparison materials for stable isotopes of light elements (1993),pp 155-162) operating on the mass 44, 45 and 46 currents.

[0171]¹³C/¹²C and ¹⁴C Isotope Characterization:

[0172] Petrochemicals have δ¹³ values of approximately −27.5‰, while C3derived sugars have δ¹³ values of −24‰ and C4 derived sugars have δ¹³values of −14‰, using the NBS standard (see Coplen et al., EOS,Transactions, American Geophysical Union 77, 27,255, (1996). On thisbasis, it was anticipated that 1,3-propanediol would have δ¹³ valuessimilar to the corn starch from which it is derived and f_(M) valuessimilar to modern carbon. Similarly, the polypropylene terephthalatederived from biosourced 1,3-propanediol will have δ¹³ values for theglycol component similar to the corn starch while the terephthaloylcomponent will be similar to petrochemicals.

[0173]¹³C and ¹⁴C data for dual isotopic characterization experiments ofthe above mentioned samples are given in Table 7, and shown graphicallyin FIG. 1. It is clear that complete discrimination has been achievedfor all samples, including those that are identical chemically. Isotopedifferences were so great, compared to the internal reproducibility,that for these particular materials either isotope would have beensufficient to make a differentiation.

[0174]¹⁴C provides the potential for “absolute” year-of-growthdiscrimination, as well as biospheric-fossil apportionment of the testmaterials, using the ¹⁴C input/decay function discussed previously. ¹³Cis interesting especially as an indicator of C3 or C4 plant originmaterial. For example, Table 7 and FIG. 1 illustrate the following:

1 1 1 12145 DNA Klebsiella pneumoniae 1 gtcgaccacc acggtggtga ctttaatgccgctctcatgc agcagctcgg tggcggtctc 60 aaaattcagg atgtcgccgg tatagtttttgataatcagc aagacgcctt cgccgccgtc 120 aatttgcatc gcgcattcaa acattttgtccggcgtcggc gaggtgaata tttcccccgg 180 acaggcgccg gagagcatgc cctggccgatatagccgcag tgcatcggtt catgtccgct 240 gccgccgccg gagagcaggg ccaccttgccagccaccggc gcgtcggtgc gggtcacata 300 cagcgggtcc tgatgcaggg tcagctgcggatgggcttta gccagcccct gtaattgttc 360 attcagtaca tcttcaacac ggttaatcagctttttcatt attcagtgct ccgttggaga 420 aggttcgatg ccgcctctct gctggcggaggcggtcatcg cgtaggggta tcgtctgacg 480 gtggagcgtg cctggcgata tgatgattctggctgagcgg acgaaaaaaa gaatgccccg 540 acgatcgggt ttcattacga aacattgcttcctgattttg tttctttatg gaacgttttt 600 gctgaggata tggtgaaaat gcgagctggcgcgctttttt tcttctgcca taagcggcgg 660 tcaggatagc cggcgaagcg ggtgggaaaaaattttttgc tgattttctg ccgactgcgg 720 gagaaaaggc ggtcaaacac ggaggattgtaagggcatta tgcggcaaag gagcggatcg 780 ggatcgcaat cctgacagag actagggttttttgttccaa tatggaacgt aaaaaattaa 840 cctgtgtttc atatcagaac aaaaaggcgaaagatttttt tgttccctgc cggccctaca 900 gtgatcgcac tgctccggta cgctccgttcaggccgcgct tcactggccg gcgcggataa 960 cgccagggct catcatgtct acatgcgcacttatttgagg gtgaaaggaa tgctaaaagt 1020 tattcaatct ccagccaaat atcttcagggtcctgatgct gctgttctgt tcggtcaata 1080 tgccaaaaac ctggcggaga gcttcttcgtcatcgctgac gatttcgtaa tgaagctggc 1140 gggagagaaa gtggtgaatg gcctgcagagccacgatatt cgctgccatg cggaacggtt 1200 taacggcgaa tgcagccatg cggaaatcaaccgtctgatg gcgattttgc aaaaacaggg 1260 ctgccgcggc gtggtcggga tcggcggtggtaaaaccctc gataccgcga aggcgatcgg 1320 ttactaccag aagctgccgg tggtggtgatcccgaccatc gcctcgaccg atgcgccaac 1380 cagcgcgctg tcggtgatct acaccgaagcgggcgagttt gaagagtatc tgatctatcc 1440 gaaaaacccg gatatggtgg tgatggacacggcgattatc gccaaagcgc cggtacgcct 1500 gctggtctcc ggcatgggcg atgcgctctccacctggttc gaggccaaag cttgctacga 1560 tgcgcgcgcc accagcatgg ccggaggacagtccaccgag gcggcgctga gcctcgcccg 1620 cctgtgctat gatacgctgc tggcggagggcgaaaaggcc cgtctggcgg cgcaggccgg 1680 ggtagtgacc gaagcgctgg agcgcatcatcgaggcgaac acttacctca gcggcattgg 1740 ctttgaaagc agtggcctgg ccgctgcccatgcaatccac aacggtttca ccattcttga 1800 agagtgccat cacctgtatc acggtgagaaagtggccttc ggtaccctgg cgcagctggt 1860 gctgcagaac agcccgatgg acgagattgaaacggtgcag ggcttctgcc agcgcgtcgg 1920 cctgccggtg acgctcgcgc agatgggcgtcaaagagggg atcgacgaga aaatcgccgc 1980 ggtggcgaaa gctacctgcg cggaaggggaaaccatccat aatatgccgt ttgcggtgac 2040 cccggagagc gtccatgccg ctatcctcaccgccgatctg ttaggccagc agtggctggc 2100 gcgttaattc gcggtggcta aaccgctggcccaggtcagc ggtttttctt tctcccctcc 2160 ggcagtcgct gccggagggg ttctctatggtacaacgcgg aaaaggatat gactgttcag 2220 actcaggata ccgggaaggc ggtctcttccgtcattgccc agtcatggca ccgctgcagc 2280 aagtttatgc agcgcgaaac ctggcaaacgccgcaccagg cccagggcct gaccttcgac 2340 tccatctgtc ggcgtaaaac cgcgctgctcaccatcggcc aggcggcgct ggaagacgcc 2400 tgggagttta tggacggccg cccctgcgcgctgtttattc ttgatgagtc cgcctgcatc 2460 ctgagccgtt gcggcgagcc gcaaaccctggcccagctgg ctgccctggg atttcgcgac 2520 ggcagctatt gtgcggagag cattatcggcacctgcgcgc tgtcgctggc cgcgatgcag 2580 ggccagccga tcaacaccgc cggcgatcggcattttaagc aggcgctaca gccatggagt 2640 ttttgctcga cgccggtgtt tgataaccacgggcggctgt tcggctctat ctcgctttgc 2700 tgtctggtcg agcaccagtc cagcgccgacctctccctga cgctggccat cgcccgcgag 2760 gtgggtaact ccctgcttac cgacagcctgctggcggaat ccaaccgtca cctcaatcag 2820 atgtacggcc tgctggagag catggacgatggggtgatgg cgtggaacga acagggcgtg 2880 ctgcagtttc tcaatgttca ggcggcgagactgctgcatc ttgatgctca ggccagccag 2940 gggaaaaata tcgccgatct ggtgaccctcccggcgctgc tgcgccgcgc catcaaacac 3000 gcccgcggcc tgaatcacgt cgaagtcacctttgaaagtc agcatcagtt tgtcgatgcg 3060 gtgatcacct taaaaccgat tgtcgaggcgcaaggcaaca gttttattct gctgctgcat 3120 ccggtggagc agatgcggca gctgatgaccagccagctcg gtaaagtcag ccacaccttt 3180 gagcagatgt ctgccgacga tccggaaacccgacgcctga tccactttgg ccgccaggcg 3240 gcgcgcggcg gcttcccggt gctactgtgcggcgaagagg gggtcgggaa agagctgctg 3300 agccaggcta ttcacaatga aagcgaacgggcgggcggcc cctacatctc cgtcaactgc 3360 cagctatatg ccgacagcgt gctgggccaggactttatgg gcagcgcccc taccgacgat 3420 gaaaatggtc gcctgagccg ccttgagctggccaacggcg gcaccctgtt tctggaaaag 3480 atcgagtatc tggcgccgga gctgcagtcggctctgctgc aggtgattaa gcagggcgtg 3540 ctcacccgcc tcgacgcccg gcgcctgatcccggtggatg tgaaggtgat tgccaccacc 3600 accgtcgatc tggccaatct ggtggaacagaaccgcttta gccgccagct gtactatgcg 3660 ctgcactcct ttgagatcgt catcccgccgctgcgcgccc gacgcaacag tattccgtcg 3720 ctggtgcata accggttgaa gagcctggagaagcgtttct cttcgcgact gaaagtggac 3780 gatgacgcgc tggcacagct ggtggcctactcgtggccgg ggaatgattt tgagctcaac 3840 agcgtcattg agaatatcgc catcagcagcgacaacggcc acattcgcct gagtaatctg 3900 ccggaatatc tcttttccga gcggccgggcggggatagcg cgtcatcgct gctgccggcc 3960 agcctgactt ttagcgccat cgaaaaggaagctattattc acgccgcccg ggtgaccagc 4020 gggcgggtgc aggagatgtc gcagctgctcaatatcggcc gcaccaccct gtggcgcaaa 4080 atgaagcagt acgatattga cgccagccagttcaagcgca agcatcaggc ctagtctctt 4140 cgattcgcgc catggagaac agggcatccgacaggcgatt gctgtagcgt ttgagcgcgt 4200 cgcgcagcgg atgcgcgcgg tccatggccgtcagcaggcg ttcgagccga cgggactggg 4260 tgcgcgccac gtgcagctgg gcagaggcgagattcctccc cgggatcacg aactgtttta 4320 acgggccgct ctcggccata ttgcggtcgataagccgctc cagggcggtg atctcctctt 4380 cgccgatcgt ctggctcagg cgggtcaggccccgcgcatc gctggccagt tcagccccca 4440 gcacgaacag cgtctgctga atatggtgcaggctttcccg cagcccggcg tcgcgggtcg 4500 tggcgtagca gacgcccagc tgggatatcagttcatcgac ggtgccgtag gcctcgacgc 4560 gaatatggtc tttctcgatg cggctgccgccgtacagggc ggtggtgcct ttatccccgg 4620 tgcgggtata gatacgatac attcagtttctctcacttaa cggcaggact ttaaccagct 4680 gcccggcgtt ggcgccgagc gtacgcagttgatcgtcgct atcggtgacg tgtccggtag 4740 ccagcggcgc gtccgccggc agctgggcatgagtgagggc tatctcgccg gacgcgctga 4800 gcccgatacc cacccgcagg ggcgagcttctggccgccag ggcgcccagc gcagcggcgt 4860 caccgcctcc gtcataggtt atggtctggcaggggacccc ctgctcctcc agcccccagc 4920 acagctcatt gatggcgccg gcatggtgcccgcgcggatc gtaaaacagg cgtacgcctg 4980 gcggtgaaag cgacatgacg gtcccctcgttaacactcag aatgcctggc ggaaaatcgc 5040 ggcaatctcc tgctcgttgc ctttacgcgggttcgagaac gcattgccgt cttttagagc 5100 catctccgcc atgtagggga agtcggcctcttttaccccc agatcgcgca gatgctgcgg 5160 aataccgata tccatcgaca gacgcgtgatagcggcgatg gctttttccg ccgcgtcgag 5220 agtggacagt ccggtgatat tttcgcccatcagttcagcg atatcggcga atttctccgg 5280 gttggcgatc aggttgtagc gcgccacatgcggcagcagg acagcgttgg ccacgccgtg 5340 cggcatgtcg tacaggccgc ccagctggtgcgccatggcg tgcacgtagc cgaggttggc 5400 gttattgaaa gccatcccgg ccagcagagaagcataggcc atgttttccc gcgcctgcag 5460 attgctgccg agggccacgg cctggcgcaggttgcgggcg atgaggcgga tcgcctgcat 5520 ggcggcggcg tccgtcaccg ggttagcgtctttggagata taggcctcta cggcgtgggt 5580 cagggcatcc atcccggtcg ccgcggtcagggcggccggt ttaccgatca tcagcagtgg 5640 atcgttgata gagaccgacg gcagtttgcgccagctgacg atcacaaact tcactttggt 5700 ttcggtgttg gtcaggacgc agtggcgggtgacctcgctg gcggtgccgg cggtggtatt 5760 gaccgcgacg ataggcggca gcgggttggtcagggtctcg attccggcat actggtacag 5820 atcgccctca tgggtggcgg cgatgccgatgcctttgccg caatcgtgcg ggctgccgcc 5880 gcccacggtg acgatgatgt cgcactgttcgcggcgaaac acggcgaggc cgtcgcgcac 5940 gttggtgtct ttcgggttcg gctcgacgccgtcaaagatc gccacctcga tcccggcctc 6000 ccgcagataa tgcagggttt tgtccaccgcgccatcttta attgcccgca ggcctttgtc 6060 ggtgaccagc agggcttttt tcccccccagcagctggcag cgttcgccga ctacggaaat 6120 ggcgttgggg ccaaaaaagt taacgtttggcaccagataa tcaaacatac gatagctcat 6180 aatatacctt ctcgcttcag gttataatgcggaaaaacaa tccagggcgc actgggctaa 6240 taattgatcc tgctcgaccg taccgccgctaacgccgacg gcgccaatta cctgctcatt 6300 aaaaataact ggcaggccgc cgccaaaaataataattcgc tgttggttgg ttagctgcag 6360 accgtacaga gattgtcctg gctggaccgctgacgtaatt tcatgggtac cttgcttcag 6420 gctgcaggcg ctccaggctt tattcagggaaatatcgcag ctggagacga aggcctcgtc 6480 catccgctgg ataagcagcg tgttgcctccgcggtcaact acggaaaaca ccaccgccac 6540 gttgatctca gtggcttttt tttccaccgccgccgccatt tgctgggcgg cggccagggt 6600 gattgtctga acttgttggc tcttgttcatcattctctcc cgcaccagga taacgctggc 6660 gcgaatagtc agtagggggc gatagtaaaaaactattacc attcggttgg cttgctttat 6720 ttttgtcagc gttattttgt cgcccgccatgatttagtca atagggttaa aatagcgtcg 6780 gaaaaacgta attaagggcg ttttttattaattgatttat atcattgcgg gcgatcacat 6840 tttttatttt tgccgccgga gtaaagtttcatagtgaaac tgtcggtaga tttcgtgtgc 6900 caaattgaaa cgaaattaaa tttatttttttcaccactgg ctcatttaaa gttccgctat 6960 tgccggtaat ggccgggcgg caacgacgctggcccggcgt attcgctacc gtctgcggat 7020 ttcacctttt gagccgatga acaatgaaaagatcaaaacg atttgcagta ctggcccagc 7080 gccccgtcaa tcaggacggg ctgattggcgagtggcctga agaggggctg atcgccatgg 7140 acagcccctt tgacccggtc tcttcagtaaaagtggacaa cggtctgatc gtcgaactgg 7200 acggcaaacg ccgggaccag tttgacatgatcgaccgatt tatcgccgat tacgcgatca 7260 acgttgagcg cacagagcag gcaatgcgcctggaggcggt ggaaatagcc cgtatgctgg 7320 tggatattca cgtcagccgg gaggagatcattgccatcac taccgccatc acgccggcca 7380 aagcggtcga ggtgatggcg cagatgaacgtggtggagat gatgatggcg ctgcagaaga 7440 tgcgtgcccg ccggaccccc tccaaccagtgccacgtcac caatctcaaa gataatccgg 7500 tgcagattgc cgctgacgcc gccgaggccgggatccgcgg cttctcagaa caggagacca 7560 cggtcggtat cgcgcgctac gcgccgtttaacgccctggc gctgttggtc ggttcgcagt 7620 gcggccgccc cggcgtgttg acgcagtgctcggtggaaga ggccaccgag ctggagctgg 7680 gcatgcgtgg cttaaccagc tacgccgagacggtgtcggt ctacggcacc gaagcggtat 7740 ttaccgacgg cgatgatacg ccgtggtcaaaggcgttcct cgcctcggcc tacgcctccc 7800 gcgggttgaa aatgcgctac acctccggcaccggatccga agcgctgatg ggctattcgg 7860 agagcaagtc gatgctctac ctcgaatcgcgctgcatctt cattactaaa ggcgccgggg 7920 ttcagggact gcaaaacggc gcggtgagctgtatcggcat gaccggcgct gtgccgtcgg 7980 gcattcgggc ggtgctggcg gaaaacctgatcgcctctat gctcgacctc gaagtggcgt 8040 ccgccaacga ccagactttc tcccactcggatattcgccg caccgcgcgc accctgatgc 8100 agatgctgcc gggcaccgac tttattttctccggctacag cgcggtgccg aactacgaca 8160 acatgttcgc cggctcgaac ttcgatgcggaagattttga tgattacaac atcctgcagc 8220 gtgacctgat ggttgacggc ggcctgcgtccggtgaccga ggcggaaacc attgccattc 8280 gccagaaagc ggcgcgggcg atccaggcggttttccgcga gctggggctg ccgccaatcg 8340 ccgacgagga ggtggaggcc gccacctacgcgcacggcag caacgagatg ccgccgcgta 8400 acgtggtgga ggatctgagt gcggtggaagagatgatgaa gcgcaacatc accggcctcg 8460 atattgtcgg cgcgctgagc cgcagcggctttgaggatat cgccagcaat attctcaata 8520 tgctgcgcca gcgggtcacc ggcgattacctgcagacctc ggccattctc gatcggcagt 8580 tcgaggtggt gagtgcggtc aacgacatcaatgactatca ggggccgggc accggctatc 8640 gcatctctgc cgaacgctgg gcggagatcaaaaatattcc gggcgtggtt cagcccgaca 8700 ccattgaata aggcggtatt cctgtgcaacagacaaccca aattcagccc tcttttaccc 8760 tgaaaacccg cgagggcggg gtagcttctgccgatgaacg cgccgatgaa gtggtgatcg 8820 gcgtcggccc tgccttcgat aaacaccagcatcacactct gatcgatatg ccccatggcg 8880 cgatcctcaa agagctgatt gccggggtggaagaagaggg gcttcacgcc cgggtggtgc 8940 gcattctgcg cacgtccgac gtctcctttatggcctggga tgcggccaac ctgagcggct 9000 cggggatcgg catcggtatc cagtcgaaggggaccacggt catccatcag cgcgatctgc 9060 tgccgctcag caacctggag ctgttctcccaggcgccgct gctgacgctg gagacctacc 9120 ggcagattgg caaaaacgct gcgcgctatgcgcgcaaaga gtcaccttcg ccggtgccgg 9180 tggtgaacga tcagatggtg cggccgaaatttatggccaa agccgcgcta tttcatatca 9240 aagagaccaa acatgtggtg caggacgccgagcccgtcac cctgcacatc gacttagtaa 9300 gggagtgacc atgagcgaga aaaccatgcgcgtgcaggat tatccgttag ccacccgctg 9360 cccggagcat atcctgacgc ctaccggcaaaccattgacc gatattaccc tcgagaaggt 9420 gctctctggc gaggtgggcc cgcaggatgtgcggatctcc cgccagaccc ttgagtacca 9480 ggcgcagatt gccgagcaga tgcagcgccatgcggtggcg cgcaatttcc gccgcgcggc 9540 ggagcttatc gccattcctg acgagcgcattctggctatc tataacgcgc tgcgcccgtt 9600 ccgctcctcg caggcggagc tgctggcgatcgccgacgag ctggagcaca cctggcatgc 9660 gacagtgaat gccgcctttg tccgggagtcggcggaagtg tatcagcagc ggcataagct 9720 gcgtaaagga agctaagcgg aggtcagcatgccgttaata gccgggattg atatcggcaa 9780 cgccaccacc gaggtggcgc tggcgtccgactacccgcag gcgagggcgt ttgttgccag 9840 cgggatcgtc gcgacgacgg gcatgaaagggacgcgggac aatatcgccg ggaccctcgc 9900 cgcgctggag caggccctgg cgaaaacaccgtggtcgatg agcgatgtct ctcgcatcta 9960 tcttaacgaa gccgcgccgg tgattggcgatgtggcgatg gagaccatca ccgagaccat 10020 tatcaccgaa tcgaccatga tcggtcataacccgcagacg ccgggcgggg tgggcgttgg 10080 cgtggggacg actatcgccc tcgggcggctggcgacgctg ccggcggcgc agtatgccga 10140 ggggtggatc gtactgattg acgacgccgtcgatttcctt gacgccgtgt ggtggctcaa 10200 tgaggcgctc gaccggggga tcaacgtggtggcggcgatc ctcaaaaagg acgacggcgt 10260 gctggtgaac aaccgcctgc gtaaaaccctgccggtggtg gatgaagtga cgctgctgga 10320 gcaggtcccc gagggggtaa tggcggcggtggaagtggcc gcgccgggcc aggtggtgcg 10380 gatcctgtcg aatccctacg ggatcgccaccttcttcggg ctaagcccgg aagagaccca 10440 ggccatcgtc cccatcgccc gcgccctgattggcaaccgt tccgcggtgg tgctcaagac 10500 cccgcagggg gatgtgcagt cgcgggtgatcccggcgggc aacctctaca ttagcggcga 10560 aaagcgccgc ggagaggccg atgtcgccgagggcgcggaa gccatcatgc aggcgatgag 10620 cgcctgcgct ccggtacgcg acatccgcggcgaaccgggc acccacgccg gcggcatgct 10680 tgagcgggtg cgcaaggtaa tggcgtccctgaccggccat gagatgagcg cgatatacat 10740 ccaggatctg ctggcggtgg atacgtttattccgcgcaag gtgcagggcg ggatggccgg 10800 cgagtgcgcc atggagaatg ccgtcgggatggcggcgatg gtgaaagcgg atcgtctgca 10860 aatgcaggtt atcgcccgcg aactgagcgcccgactgcag accgaggtgg tggtgggcgg 10920 cgtggaggcc aacatggcca tcgccggggcgttaaccact cccggctgtg cggcgccgct 10980 ggcgatcctc gacctcggcg ccggctcgacggatgcggcg atcgtcaacg cggaggggca 11040 gataacggcg gtccatctcg ccggggcggggaatatggtc agcctgttga ttaaaaccga 11100 gctgggcctc gaggatcttt cgctggcggaagcgataaaa aaatacccgc tggccaaagt 11160 ggaaagcctg ttcagtattc gtcacgagaatggcgcggtg gagttctttc gggaagccct 11220 cagcccggcg gtgttcgcca aagtggtgtacatcaaggag ggcgaactgg tgccgatcga 11280 taacgccagc ccgctggaaa aaattcgtctcgtgcgccgg caggcgaaag agaaagtgtt 11340 tgtcaccaac tgcctgcgcg cgctgcgccaggtctcaccc ggcggttcca ttcgcgatat 11400 cgcctttgtg gtgctggtgg gcggctcatcgctggacttt gagatcccgc agcttatcac 11460 ggaagccttg tcgcactatg gcgtggtcgccgggcagggc aatattcggg gaacagaagg 11520 gccgcgcaat gcggtcgcca ccgggctgctactggccggt caggcgaatt aaacgggcgc 11580 tcgcgccagc ctctctcttt aacgtgctatttcaggatgc cgataatgaa ccagacttct 11640 accttaaccg ggcagtgcgt ggccgagtttcttggcaccg gattgctcat tttcttcggc 11700 gcgggctgcg tcgctgcgct gcgggtcgccggggccagct ttggtcagtg ggagatcagt 11760 attatctggg gccttggcgt cgccatggccatctacctga cggccggtgt ctccggcgcg 11820 cacctaaatc cggcggtgac cattgccctgtggctgttcg cctgttttga acgccgcaag 11880 gtgctgccgt ttattgttgc ccagacggccggggccttct gcgccgccgc gctggtgtat 11940 gggctctatc gccagctgtt tctcgatcttgaacagagtc agcatatcgt gcgcggcact 12000 gccgccagtc ttaacctggc cggggtcttttccacgtacc cgcatccaca tatcactttt 12060 atacaagcgt ttgccgtgga gaccaccatcacggcaatcc tgatggcgat gatcatggcc 12120 ctgaccgacg acggcaacgg aattc 12145

What is claimed is:
 1. A product of a process comprising thebioconversion of a fermentable carbon substrate, other than glycerol ordihydroxyacetone, to 1,3-propanediol by a single microorganism having atleast one gene that expresses a dehydratase enzyme by contacting saidmicroorganism with the carbon substrate.
 2. 1,3-Propanediol produced bya process comprising the steps of: (i) contacting a medium containing atleast one fermentable carbon substrate with a single microorganism toyield a culture medium, the at least one carbon substrate is selectedfrom the group consisting of monosaccharides, oligosaccharides, andpolysaccharides, provided that the carbon substrate is other thanglycerol or dihydroxyacetone, and the single microorganism is selectedfrom the group consisting of members of the genera Klebsiella,Citrobacter, recombinant Escherichia, or is a recombinant microorganismtransformed with a gene encoding a diol dehydratase enzyme or a glyceroldehydratase enzyme, (ii) incubating the culture medium of step (i) undersuitable conditions to produce 1,3-propanediol; and (iii) recovering1,3-propanediol from the culture medium.
 3. 1,3-Propanediol produced bya process comprising the steps of: (i) contacting a culture mediumcontaining glucose with a recombinant Escherichia coli transformed witha gene encoding a diol dehydratase enzyme or a glycerol dehydrataseenzyme; (ii) incubating the culture medium of step (i) under suitableconditions to produce 1,3-propanediol; and recovering 1,3-propanediolfrom the culture medium.
 4. 1,3-Propanediol produced by the process ofclaim 1 having a δ¹³C of about −10.9 to about −15.4 and a f_(M) ¹⁴C ofabout 1.04 to about 1.18.
 5. 1,3-Propanediol produced by the process ofclaim 4 having a δ¹³C of about −13.22 to about −14.54 and a f_(M) ¹⁴C ofabout 1.106 to about 1.129.
 6. 1,3-Propanediol produced by the processof claim 4 having a δ¹³C of about −13.84 to about −13.92, and a f_(M)¹⁴C of about 1.111 to about 1.124.
 7. A polymer comprising at least tworepeating units of biosourced 1,3-propanediol characterized by a δ¹³C ofabout −10.74 to about −17.02 and a f_(M) ¹⁴C of about 1.003 to about1.232.
 8. A polymer comprising at least two repeating units ofbiosourced 1,3-propanediol characterized by a δ¹³C of about −13.22 toabout −14.54 and a f_(M) ¹⁴C of about 1.106 to about 1.129.
 9. A polymercomprising at least two repeating units of biosourced 1,3-propanediolcharacterized by a δ¹³C of about −13.84 to about −13.92, and a f_(M) ¹⁴Cof about 1.111 to about 1.124.
 10. A polymer comprising at least tworepeating units of biosourced polypropylene terephthalate characterizedby a δ¹³C of about −23.76 to about −25.85, and a f_(M) ¹⁴C of about0.241 to about 0.373.
 11. A polymer comprising at least two repeatingunits of biosourced polypropylene terephthalate, said polypropyleneterephthalate having δ¹³C of about −24.50 to about −25.07, and a f_(M)¹⁴C of about 0.286 to about 0.326.
 12. A polymer comprising at least tworepeating units of biosourced polypropylene terephthalate characterizedby a δ¹³C of about −24.74 to about. −24.88, and a f_(M) ¹⁴C of about0.299 to about 0.309.
 13. A polymer comprising at least two repeatingunits of biosourced polypropylene terephthalate, said polypropyleneterephthalate having δ¹³C of about −24.75 to about −24.82, and a f_(M)¹⁴C of about 0.303 to about 0.309.
 14. A co-polymer comprising a blendof two or more polymers, at least one of the polymers comprisingbiosourced 1,3-propanediol characterized by a δ¹³C of about −13.84 toabout −13.92 and a f_(M) ¹⁴C of about 1.109 to about 1.126.
 15. Anarticle of manufacture comprising the composition of any one of claims4, 7, 10 or 14 in a form selected from the group consisting of a film, afiber, a particle, a package, and~a molded article.
 16. A method foridentifying the presence of a biosourced 1,3-propanediol in a sample,the method comprising (a) purifying the 1,3-propanediol from the sample;(b) determining the δ¹³C and f_(M) ¹⁴C characterizing the sample of step(a), wherein a δ¹³C of about −10.9 to about −15.4 and a f_(M) ¹⁴C ofabout 1.04 to about 1.18 indicates the presence of a bio-sourced1,3-propanediol.